Conjugates for treating neurodegenerative diseases and disorders

ABSTRACT

A conjugate comprising L-DOPA covalently linked to at least one γ-aminobutyric acid (GABA) moiety, an ester and/or an addition salt thereof are disclosed, as well as uses thereof for treating a neurodegenerative disease or disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/867,055 filed on Oct. 28, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2009/000158 having International filingdate of Feb. 11, 2009, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/064,017 filed on Feb. 11, 2008.The contents of the above Applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to novelcompounds, to pharmaceutical compositions containing same and to usesthereof in the treatment of neurodegenerative diseases and disorders,such as Parkinson's disease.

Parkinson's disease is an age-related disorder characterized byprogressive loss of dopamine producing neurons in the substantia nigraof the midbrain, which in turn leads to progressive loss of motorfunctions manifested through symptoms such as tremor, rigidity andataxia. Parkinson's disease can be treated by administration ofpharmacological doses of the precursor of dopamine, L-DOPA (Marsden,Trends Neurosci. 9:512, 1986; Vinken et al., in Handbook of ClinicalNeurology p. 185, Elsevier, Amsterdam, 1986). Although such treatment iseffective in early stage Parkinson's patients, progressive loss ofsubstantia nigra cells eventually leads to an inability of remainingcells to synthesize sufficient dopamine from the administered precursorand to diminishing pharmacogenic effect.

Recently, Neurologix Inc. announced interim results of a gene therapyclinical trial for patients with Parkinson's disease. The gene therapyinvolved transforming target brain cells with glutamic aciddecarboxylase (GAD) gene to thereby increase GABA synthesis in thebrain. According to the interim report(wwwdotbiologynewsdotnet/archives/2005/09/25/neurologix_announces_positive_results_of_gene_therapy_clinical_trial_in_parkinsons_diseasedothtml),treated Parkinson's disease patients exhibited statistically significantimprovement in motor function and a strong trend toward improvement ofactivities of daily living.

Unfortunately, clinical use of GABA for treating neurodegenerativedisorders is presently limited since the GABA molecule compriseshydrophilic functional groups (e.g., a free carboxylic acid group and afree amino group) and therefore does not effectively cross the bloodbrain barrier (BBB).

In an attempt to overcome the limitations associated with theadministration of GABA to the brain, Prof. Nudelman and co-researchers,which are co-inventors of the present invention, have designed andsuccessfully practiced a series of conjugates of psychotropic drugs andGABA. These conjugates and their advantageous use in the treatment ofpsychotic and/or proliferative diseases and disorders are described indetail in International Patent Application published as WO 03/026563,which is incorporated by reference as if fully set forth herein.

Accordingly, International Patent Application WO 2005/092392, by thesame inventors, which is also incorporated by reference as if fully setforth herein, teach psychotropic drugs coupled to GABA.

SUMMARY OF THE INVENTION

The present invention, in some embodiments thereof, provides novelconjugates of L-DOPA and GABA; which can be used safely and effectivelyin treating neurodegenerative disorders, such as Parkinson's disease.

According to one aspect of embodiments of the invention there isprovided a conjugate comprising L-DOPA covalently linked to at least oneγ-aminobutyric acid (GABA) moiety. The conjugate can be in a form of apharmaceutically acceptable salt thereof.

According to another aspect of embodiments of the invention there isprovided a pharmaceutical composition comprising, as an activeingredient, a conjugate comprising L-DOPA covalently linked to at leastone GABA moiety and a pharmaceutically acceptable carrier.

According to yet another aspect of embodiments of the invention there isprovided an article-of-manufacturing comprising a pharmaceuticalcomposition which comprises, as an active ingredient, a conjugatecomprising L-DOPA covalently linked to at least one GABA moiety and apharmaceutically acceptable carrier, the composition being packaged in apackaging material and identified in print, on or in the packagingmaterial, for use in the treatment of a neurodegenerative disease ordisorder

According to still another aspect of embodiments of the invention thereis provided a method of treating a neurodegenerative disease ordisorder. The method is effected by administering to a subject in needthereof a therapeutically effective amount of a conjugate comprisingL-DOPA covalently linked to at least one GABA moiety, thereby treatingthe neurodegenerative disease or disorder disease.

According to an additional aspect of embodiments of the invention thereis provided use of a conjugate comprising L-DOPA covalently linked to atleast one GABA moiety in the preparation of a medicament.

According to some embodiments the medicament is for treating aneurodegenerative disease or disorder.

According to some embodiments of the invention described below, theconjugate comprises a single GABA moiety linked to L-DOPA.

According to some embodiments, the conjugate comprises two GABA moietieslinked to L-DOPA.

According to some embodiments, the conjugate comprises three GABAmoieties linked to L-DOPA.

According to some embodiments, the L-DOPA and each of GABA moieties arelinked therebetween via a covalent bond selected from the groupconsisting of a carboxylic ester bond, an alkyloxy carboxylic ester bondand an amide bond.

According to some embodiments, the covalent bond is an amide bond and aGABA moiety is linked to an amine functional group of L-DOPA (whennon-conjugated).

According to some embodiments, the covalent bond is an ester bond and aGABA moiety is linked to one or both hydroxy groups of L-DOPA (whennon-conjugated).

According to some embodiments, the covalent bond is an alkyloxycarboxylic ester bond and a GABA moiety is linked to the carboxylic acidgroup of L-DOPA (when non-conjugated).

According to some embodiments, the neurodegenerative disease or disorderis Parkinson's disease.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 presents a schematic representation of the protocol used in theexperiments described in Example 2 hereinbelow for examining theprotective effect of BL-1023, an L-DOPA-GABA conjugate according to someembodiments of the invention, in a MPTP acute toxicity model. Mice wereadministered 4 injections IP of MPTP (each injection contained 20 mg/kg,5 ml/kg) or saline alone (control) at 2 hours intervals on day 0. Micewere then administered subcutaneously test solutions (TI) (eithersaline, L-DOPA or BL-1023) once daily throughout the 8 successivetreatment days (days 0-7). On day 7, the mice were sacrificed and theirbrains were removed for immunohistochemistry analysis of the number oftyrosine hyroxylase immunostained cells at the level of the SubstantiaNigra (SNpc).

FIGS. 2(A-H) present comparative plots showing the data obtained in anopen field test for examining the protective effect of AN-490, anL-DOPA-GABA conjugate according to some embodiments of the invention, ina MPTP acute toxicity model. Mice were administered the followingtreatments: saline (filled red circles connected with a dashed red line,Control), L-DOPA 25 mg/kg (filled black triangles connected with adashed black line, L-DOPA), AN-490 67.5 mg/kg (filled green squaresconnected with a dashed green line, AN-490), MPTP (empty blue circlesconnected with a blue line, MPTP), MPTP+L-DOPA 25 mg/kg (empty blacktriangles connected with a black line, MPTP+DOPA) and MPTP+AN-490 67.5mg/kg (filled green squares connected with a green line, MPTP+AN-490).The MPTP neurotoxin was administered on day 0, with 4 IP injections(each injection contained 20 mg/kg, 5 ml/kg) in saline. The varioustreatments or saline were administered on days 0, 1, 2, 3, 6, 7, 13 and16. Each group of mice (n=6) was subjected, on days 3, 6, 8 and 16, tothe open field test, and the following parameters were scored: thedistance moved (FIGS. 2A and 2B), velocity (FIGS. 2C and 2D), time spentimmobile (FIGS. 2E and 2F) and time spent in a high level of mobility(FIGS. 2G and 2H; strong mobility) during a period of 20 minutes. FIGS.2A, 2C, 2E and 2G present the data obtained for the control mice, micetreated with L-DOPA and mice treated with AN-490. FIGS. 2B, 2D, 2F and2H present the data obtained for control mice or mice treated with MPTP,MPTP with L-DOPA and MPTP with AN-490. The group of mice which receivedthe MPTP+AN-490 exhibited the highest velocity (FIG. 2B), was highlymobile (FIG. 2H) and was immobile for a short time (FIG. 2F) as comparedto the other tested groups.

FIGS. 3(A-C) present bar graphs showing the RotaRod test resultsobtained for examining the protective effect of AN-490, an L-DOPA-GABAconjugate according to some embodiments of the invention, in a MPTPacute toxicity model. Mice were administered the following treatments:saline (Control), L-DOPA 25 mg/kg, AN-490 67.5 mg/kg, MPTP, MPTP+L-DOPA25 mg/kg, and MPTP+AN-490 67.5 mg/kg. The MPTP neurotoxin wasadministered on day 0, with 4 IP injections (each injection contained 20mg/kg, 5 ml/kg) in saline. The various treatments or saline wereadministered on days 0, 1, 2, 3, 6, 7, 13 and 16. Each group of mice(n=6) was subjected, on days 1, 3 and 6, to the RotaRod test. Each mousewas tested 3 times and the average value for the performance (averageduration on rod i.e. latency) of each group on day 1 (FIG. 3A), 3 (FIG.3B) and 6 (FIG. 3C) are presented.

FIG. 4 presents comparative plots showing the change in mice weightduring the MPTP acute toxicity model (as presented in Experiment 3),following treatment with: saline (filled red circles connected with adashed red line, Control), L-DOPA 25 mg/kg (filled black trianglesconnected with a dashed black line, L-DOPA), AN-490 67.5 mg/kg (filledblack squares connected with a dashed black line, AN-490), MPTP (emptyblue circles connected with a blue line, MPTP), MPTP+L-DOPA 25 mg/kg(empty black triangles connected with a black line, MPTP+DOPA),MPTP+AN-490 67.5 mg/kg (filled green squares connected with a greenline, MPTP+AN-490).

FIG. 5 presents a bar graph showing the RotaRod test results obtainedfor examining the protective effect of AN-490, an L-DOPA-GABA conjugateaccording to some embodiments of the invention, in a MPTP sub-acutetoxicity model. Mice were administered the following treatments: saline(Control), MPTP+saline (MPTP), MPTP+GABA.HCl 18.4 mg/kg (MPTP+GABA),MPTP+L-DOPA 30 mg/kg (MPTP+L-DOPA), MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg(MPTP-Ga+Do) and MPTP+AN-490 81 mg/kg (MPTP+AN-490). The MPTP neurotoxinwas administered on days 0, 1, 2, 3, 4 and 5 as one IP injection of MPTPat a dose of 20 mg/kg. The various treatments or saline wereadministered starting from day 13, every day for 6 consecutive days andin the following week. Treatment was then reduced to 3 days a week. Eachgroup of mice (n=6) was then subjected, on day 24, to the RotaRod testand the data observed for the average duration on Rod, tested threetimes, are presented.

FIG. 6 presents comparative plots showing the change in mice weightduring the MPTP sub-acute toxicity model (Experiment 3), followingtreatment with: saline (blue filled diamonds, Control), MPTP+saline(pink filled squares, MPTP), MPTP+GABA.HCl 18.4 mg/kg (black filledtriangles), MPTP+L-DOPA 30 mg/kg (empty red squares), MPTP+L-DOPA 30mg/kg+GABA 18.4 mg/kg (purple cross) and MPTP+AN-490 81 mg/kg (browncircle).

FIG. 7 presents a bar graph showing the observed vitality signs of miceduring the MPTP sub-acute toxicity model (Experiment 3), as describedfor FIG. 6 hereinabove. Six independent observers estimated the level ofmice vitality by the level of motion of the mice (light purple, markedvitality) and fur appearance (dark purple). The results are an averageof the observers' reports.

FIGS. 8(A-F) present representative microphotographs of tyrosinehydroxylase immunostaining of the striatum and substantia-nigra of mice,showing the protective effect of AN-490 against MPTP neurotoxicity, inthe MPTP sub-acute toxicity model (Experiment 3). Shown aremicrophotograph of the striatum and substantia-nigra of mice receivingsaline (FIG. 8A, Naive), MPTP+saline (FIG. 8B, MPTP) MPTP+GABA.HCl 18.4mg/kg (FIG. 8C, MPTP+GABA), MPTP+L-DOPA 30 mg/kg (FIG. 8D, MPTP+DOPA),MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg (FIG. 8E, MPTP+Dopa+GABA) andMPTP+AN-490 81 mg/kg (FIG. 8F, MPTP+AN490). Paraffin-embedded horizontalsections of the stratium and substantia nigra were stained withhematoxylan and Tyrosine Hyroxylase antibodies. The magnification is×100 and in the insert ×200.

FIGS. 9(A-B) present bar graphs showing the RotaRod test resultsobtained for examining the protective effect of BL-1023* (denoted 1023),an L-DOPA-GABA conjugate according to some embodiments of the invention,in a MPTP acute toxicity model (Experiment 3). Mice were administeredthe following treatments: saline (Control), MPTP+saline (MPTP),MPTP+L-DOPA 30 mg/kg (MPTP+Dopa), MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg(MPTP+G+D) MPTP+BL1023* 48.4 mg/kg (MPTP+1023 30) and MPTP+BL-1023* 24.2mg/kg (MPTP+BL1023 15). The MPTP neurotoxin was administered on day 0,with 4 IP injections (each injection contained 20 mg/kg, 5 ml/kg) insaline. The various treatments or saline were administered on day 7, 9,11 and 13 (total of 4 treatments). Each group of mice (n=10) wassubjected to the RotaRod test, on day 6 (before initiation of treatment,FIG. 9A) and day 12 (after initiation of treatment, FIG. 9B) and theobserved average duration on Rod, tested three times, are presented. Theresults show that while MPTP administration led to a reduction in themice duration on the Rod (#p<0.05), BL-1203* administration, at a doseof 48.4 mg/kg, was able to reverse the observed MPTP-dependent reduction(*p<0.05).

FIGS. 10(A-B) present bar graphs showing data obtained in an open fieldtest for examining the protective effect of BL-1023* (denoted 1023), anL-DOPA-GABA conjugate according to some embodiments of the invention, ina MPTP acute toxicity model (Experiment 3). Mice were administered thefollowing treatments: saline (Control), MPTP+saline (MPTP), MPTP+L-DOPA30 mg/kg (MPTP+Dopa), MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg (MPTP+G+D),MPTP+BL1023* 48.4 mg/kg (MPTP+1023 30) and MPTP+BL-1023* 24.2 mg/kg(MPTP+BL1023 15). The MPTP neurotoxin was administered on day 0, with 4IP injections (each injection contained 20 mg/kg, 5 ml/kg) in saline.The various treatments or saline were administered on day 7, 9, 11 and13 (total of 4 treatments) and the measured velocity (FIG. 10A) anddistance moved (FIG. 10B) were measured on day 13.

FIG. 11 presents comparative plots showing the change in mice weightduring MPTP acute toxicity model (Experiment 3), following treatmentwith: saline (black filled diamonds, Control), MPTP+saline (red filledsquares, MPTP), MPTP+L-DOPA 30 mg/kg (filled blue triangles),MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg (pink filled circles), MPTP+BL1023*48.4 mg/kg (purple filled squares, denoted as MPTP+BL1023 30), andMPTP+BL-1023* 24.2 mg/kg (brown circles, denoted as MPTP+BL1023 15).

FIGS. 12(A-C) present bar graphs showing the effect of BL-1023*administration on the level of the following catecholamines:norepinephrin (FIG. 12A), dopamine (FIG. 12B) and L-DOPA (FIG. 12C) inmice. Mice were administered the following treatments: saline (Con),MPTP+saline (MPTP), MPTP+L-DOPA 30 mg/kg (denoted as MPTP+1 Dopa),MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg (denoted as MPTO+Do+GABA) andMPTP+BL1023* (MPTP+BL1023). The MPTP neurotoxin was administered on day0, with 4 IP injections (each injection contained 20 mg/kg, 5 ml/kg) insaline. The various drugs or saline were administered on day 7, 9, 11and 13 (total of 4 treatments). Brains (whole brains) of three mice fromeach group were dissected out on day 14-15 and the catecholamines levelswere determined by HPLC, according to the protocol described in theExamples section hereinbelow. Protein content of each sample wasdetermined and the level of the catecholamines was normalized to μgprotein. A significant reduction in norepinephrin and dopamine levels orenhancement of L-DOPA levels, as compared to control, is marked by*(p<0.05).

FIG. 13 presents a bar graph showing the effect of BL-1023*administration on the neuronal density in the stratium, as assessed fromTyrosine Hydroxylase staining. Mice were administered the followingtreatments: saline (control), MPTP+saline (MPTP), MPTP+L-DOPA 30 mg/kg(MPTP+L-DOPA), MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg (MPTP+L-DOPA+GABA),MPTP+BL1023* 48.4 mg/kg (denoted MPTP+1023 30) and MPTP+BL-1023* 24.2mg/kg (denoted MPTP+BL1023 15). The MPTP neurotoxin was administered onday 0, with 4 IP injections (each injection contained 20 mg/kg, 5 ml/kg)in saline. The various treatments or saline were administered on day 7,9, 11 and 13 (total of 4 treatments). Three mice, from each treatmentgroup, and four mice from the control group, were sacrificed on day 15and subjected to immunohystochemistry for tyrosine hydroxylase of thestratium according to the protocol described in the Examples sectionhereinbelow. The intensity of Tyrosine Hyroxylase (TH) staining, in thestriatum of the treated mice is presented as the average of IntensityOptical Density (IOD) and SE obtained for the total stained area.

FIG. 14 presents electronic pictures of the Immunohystochemistry (IHC)horizontal sections from the stratium of the mice treated as describedin FIG. 13, at ×40, ×100 and ×200 magnification, using ImageProsoftware.

FIG. 15 presents a bar graph showing the effect of BL-1023*administration on the neuronal density in the substantia nigra, asassessed from Tyrosine Hydroxylase staining. Mice were administered thefollowing treatments: saline (control), MPTP+saline (MPTP), MPTP+L-DOPA30 mg/kg (MPTP+L-DOPA), MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg(MPTP+L-DOPA+GABA), MPTP+BL1023* 48.4 mg/kg (MPTP+1023 30) andMPTP+BL-1023* 24.2 mg/kg (MPTP+BL1023 15). The MPTP neurotoxin wasadministered on day 0, with 4 IP injections (each injection contained 20mg/kg, 5 ml/kg) in saline. The various treatments or saline wereadministered on day 7, 9, 11 and 13 (total of 4 treatments). Three mice,from each treatment group, and four mice from the control group, weresacrificed on day 15 and subjected to immunohystochemistry for tyrosinehydroxylase of the substantia nigra according to the protocol describedin the Examples section hereinbelow. The intensity of TyrosineHyroxylase (TH) staining, in the substantia nigra of the treated mice ispresented as the number of TH stained cells and SE obtained for thetotal stained area.

FIG. 16 presents electronic pictures of the Immunohystochemistry (IHC)horizontal sections from the substantia nigra of the mice treated asdescribed in FIG. 15, at ×40, ×100 and ×200 magnification, usingImagePro software.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to novelcompounds, to pharmaceutical compositions containing same and to usesthereof in the treatment of neurodegenerative diseases and disorders,such as Parkinson's disease.

The present invention, in some embodiments thereof, is of conjugatescomprising L-DOPA covalently linked to at least one γ-aminobutyric acid(GABA) moiety, and of esters and acid addition salts of such conjugates.These conjugates are designed to have BBB permeability, and are capableto dissociate in the brain so as to release L-DOPA and GABA moieties.These conjugates therefore combine the beneficial therapeutic effects ofL-DOPA and GABA in treating neurodegenerative diseases and disorders,while facilitating the BBB permeability of these agents, which isotherwise limited (as in the case of GABA) or non-selective (as in thecase of L-DOPA).

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Neurodegenerative disorders, such as Parkinson's disease, arecharacterized by loss of neuronal functions. Presently, Levodopa, whichis also referred to herein and in the art as L-DOPA(3,4-dihydroxy-L-phenylalanine), is the most effective commerciallyavailable drug for the treatment of the symptoms of Parkinson's. L-DOPAis used as a prodrug for increasing dopamine levels, since it is capableof crossing the blood-brain barrier whereas dopamine itself cannot. OnceL-DOPA has entered the central nervous system (CNS), it is metabolizedto dopamine by aromatic-L-amino-acid decarboxylase. However, conversionto dopamine also occurs in the peripheral tissues, thereby decreasingthe available dopamine to the CNS.

While reducing the present invention to practice, the present inventorshave devised and successfully practiced novel synthetic pathways forpreparing various conjugates of L-DOPA and one or more GABA moieties.These synthetic pathways were designed such that regioselective couplingof one or more GABA moieties to L-DOPA is effected.

The present inventors have further surprisingly and unexpectedlyuncovered that these conjugates were superior to L-DOPA, whenadministered per se, when tested in the well-known mice MPTP model forParkinson's Disease (see, the Examples section that follows).

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is an effectivedopaminergic neurotoxin that causes permanent symptoms of Parkinson'sdisease in mice and is used to study the disease. As demonstrated in theExamples section that follows, L-DOPA-GABA conjugates according toembodiments of the invention were able to reduce MPTP relatedneurotoxicity. Specifically, in mice subjected to MPTP neurotoxicityaccording to a MPTP acute toxicity protocol (4 imp. injections on firstday of the experiment), the neuronal density (measured using tyrosinehydroxylase immunostaining) in the substantia nigra of mice treated withCompound 5 (BL-1023), a conjugate according to some embodiments of theinvention, was substantially higher than that in mice treated only withL-DOPA or non-treated mice (see, Table 4). Furthermore, in micesubjected to MPTP neurotoxicity according to a MPTP acute toxicityprotocol, the protective effect of Compound 21 (AN-490), anotherconjugate according to some embodiments of the invention, against MPTPneurotoxicity could be deduced from the significant increase inmotility, examined using the open field test (namely, the observedstrong mobility, and decrease in immobility) of the mice treated withCompound 21 as compared to non-treated mice (see FIG. 2). The protectiveeffect of Compound 21 was also observed by the enhanced neuronal density(measured using tyrosine hydroxylase immunostaining) in the substantianigra of mice subjected to MPTP neurotoxicity (this time in a MPTPsub-acute protocol, i.e. mice receive one MPTP injection per day forfive consecutive days), as compared to control non-treated mice (see,FIG. 8). The protective effect of Compound 16 (BL-1023*), anotherconjugate according to some embodiments of the invention, against MPTPneurotoxicity was also shown (in an acute toxicity protocol), with animproved level of performance of mice receiving Compound 16 treatment,in the RotaRod test, as compared to non-treated mice (see, FIG. 10). Itwas further shown that administration of Compound 16 to MPTP-treatedmice was able to reverse the MPTP dependent reduction in neuronaldensity in the substantia nigra and stratium (see, FIGS. 13-16).

Thus, according to one aspect of embodiments of invention there isprovided a chemical conjugate comprising L-DOPA covalently linked to atleast one γ-aminobutyric acid (GABA).

The term “GABA” or “GABA moiety”, as used herein, refers to a radical ofthe compound 4-amino-butyric acid (γ-aminobutyric acid). In the contextof the present embodiments, GABA moiety is a 4-amino-butyryl moiety, ora —(C═O)—(CH₂)₃—NH₂ group, namely a moiety which is linked to afunctional group of L-DOPA via its carbonyl carbon atom.

The amino group of a GABA moiety can be ionized at certain pH levels,depending on the conditions it is found in.

In some embodiments, L-DOPA (3,4-dihydroxy-L-phenylalanine) iscovalently linked to one GABA moiety. In some embodiments, L-DOPA iscovalently linked to two GABA moieties. In some embodiments, L-DOPA iscovalently linked to three GABA moieties, and can also be covalentlylinked to four GABA moieties.

As used herein, the term “moiety” refers to a compound having apharmacological activity. When described in the context of theconjugates presented herein, this term is understood to include a majorportion of a molecule which is covalently linked to another molecule,preferably while maintaining the activity of the molecule.

In some embodiments, GABA is coupled to L-DOPA via a covalent bond orany other bond selected or designed capable of dissociating followingcrossing of the blood-brain barrier.

Thus, in some embodiments, the covalent bond linking L-DOPA and the atleast one GABA moiety is selected or designed such that (i) it is notsusceptible to dissociation (e.g., by enzymatic reactions) in theperiphery and hence the conjugate remains substantially intact beforecrossing the BBB; and (ii) it is susceptible to dissociation in braintissues (e.g., by brain derived enzymes), and hence the conjugatedissociates following crossing the BBB, thus releasing the biologicallyactive L-DOPA and GABA.

A suitable bond can be, for example, a carboxylic ester bond, anoxyalkyl carboxylic ester bond or an amide bond, all of which can bedissociated by brain derived enzymes (e.g., brain derived esterases oramidases).

As used herein, a “carboxylic ester bond” describes an “—O—C(═O)—” bond.

As used herein, an “oxyalkyl carboxylic ester bond” describes an“O—R—O—C(═O)—” bond, where R is an alkylene, as defined hereinbelow.

An “amide bond” describes a “—NR′—C(═O)—” bond, where R′ is hydrogen,alkyl, cycloalkyl or aryl, as defined hereinbelow.

As used herein, the term “alkyl” describes a saturated aliphatichydrocarbon chain including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever anumerical range, e.g., “1-20”, is stated herein, it means that thegroup, in this case the alkyl group, may contain 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Insome embodiments, the alkyl is a medium size alkyl having 1 to 10 carbonatoms. In some embodiments, the alkyl has 1 to 4 carbon atoms.

The term “alkylene” describes an alkyl group that is linked to two othergroups. Thus, the term ethylene, for example, describes a —CH₂CH₂—group. The term “methylene” describes a —CH₂— group.

As used herein, the term “cycloalkyl” describes an all-carbon monocyclicor fused ring (i.e., rings which share an adjacent pair of carbon atoms)group wherein one of more of the rings does not have a completelyconjugated pi-electron system. Examples, without limitation, ofcycloalkyl groups include cyclopropane, cyclobutane, cyclopentane,cyclopentene, cyclohexane, cyclohexadiene, cycloheptane,cycloheptatriene and adamantane.

As used herein, the term “aryl” describes an all-carbon monocyclic orfused-ring polycyclic (i.e., rings which share adjacent pairs of carbonatoms) groups having a completely conjugated pi-electron system.Examples, without limitation, of aryl groups include phenyl,naphthalenyl and anthracenyl.

According to some embodiments of the present invention, the conjugatehas the following structure:

This conjugate is referred to herein, interchangeably, as Compound 5 orBL 1023.

The conjugates presented herein can be readily prepared by reacting GABA(optionally in a molar excess) with L-DOPA. The reaction can beperformed in the presence of a base and optionally further in thepresence of a dehydrating agent.

In some embodiments, prior to the reacting, the amine group of the GABAis protected by any of the conventional N-protecting groups (e.g., BOC).Thus, in some embodiments, prior to the reacting, GABA is converted toN-protected GABA. Similarly, in some embodiments, the carboxylic acid ofL-DOPA is protected by converting it to an ester thereof, such as, forexample, a methyl ester or a butyl ester.

The N-protected GABA and the optionally protected L-DOPA are thenreacted in the presence of a base and optionally a dehydrating agent. Inone example, the base is N-ethyldiisopropylamine and the dehydratingagent is carbonyl diimidazole (CDI). Optionally, the base istriethylamine (TEA). In some embodiments, the reaction is performed inthe presence of a solvent, preferably an organic solvent such as, forexample, dichloromethane.

Since GABA can react with various functional groups of L-DOPA (e.g., theα-amine group of L-DOPA, the para-hydroxyl group and the meta-hydroxylgroup), typically a conjugate comprising two or more GABA moietiescovalently linked to L-DOPA is obtained. As exemplified in the Examplessection that follows, a mixture of geometrical isomers (regioisomers) ofsuch a conjugate is typically obtained (see,

Scheme 1 below).

In cases where a conjugate that comprises two or more GABA moietiescovalently linked to L-DOPA is prepared, following the reacting, theprotecting groups are removed, to thereby obtain the desired product.

In cases where a conjugate that comprises a single GABA moietycovalently linked to L-DOPA is prepared, removal of the other GABAmoieties that are attached to L-DOPA can be performed. Such a removal ispreferably effected under conditions that allow selective removal ofGABA moieties, according to the desired final product.

In one example, removal of a GABA moiety is effected in the presence ofa base (e.g., sodium hydroxide), preferably in an aqueous alcoholicenvironment, so as to obtain a conjugate in which a single N-protectedGABA molecule is attached to the α-amine group of L-DOPA. Followingremoval of the N-protecting group an L-DOPA-GABA conjugate is obtained(see, Compound 5 in

Scheme 1 hereinbelow).

In some embodiments, the functional groups of L-DOPA are protected, bymeans of protecting groups, such that the reaction with N-protected GABAis effected selectively, at the desired position. In these embodiments,the protecting groups and the order of their removal are selected so asto obtain the desired product, as exemplified in the Examples sectionthat follows.

In some embodiments, when the GABA moiety is conjugated to thecarboxylic acid functional group of L-DOPA, as detailed hereinbelow, allthe functional groups of L-DOPA are first protected, via a certainorder, so as to enable a selective reaction of GABA with the carboxylicacid moiety.

The final product and the intermediates can be purified by any techniquewell known in the art (e.g., column chromatography, crystallization), asexemplified in the Examples section that follows.

Using the above procedures, the conjugates described herein aretypically obtained as HCl salts thereof. As demonstrated in the Examplessection that follows, highly pure, stable, HCl salt of an L-DOPA-GABAconjugate can be obtained.

The HCl salts, however, can be converted, via reactions well-known inthe art, to other acid addition salts of the conjugates, as detailedhereinbelow and is exemplified in the Examples section that follows.

Suitable processes of synthesizing Compound 5 (or BL-1023), including ascaled-up process, are described in details in Example 1 hereinbelow.Suitable processes for preparing other conjugates according toembodiments of the invention are also described in great detail in theExamples section the follows.

According to some embodiment of the present invention, L-DOPA isconjugated to more than one GABA moieties, and further according toother embodiments, the conjugates presented herein are L-DOPA esterderivatives, namely the carboxylic group in the L-DOPA moiety is anester.

According to some embodiments, the conjugate presented herein is anL-DOPA butyl ester linked via an amide bond to a single GABA moiety, andhaving the structure presented below (also referred to herein,interchangeably, as Compound 16 or BL-1023*):

According to some embodiments, the conjugate presented herein is anL-DOPA butyl ester linked via the hydroxyls on the benzene to two GABAmoieties, and having the structure presented below (also referred toherein, interchangeably, as Compound 21 or AN-490):

According to some embodiments, the conjugate presented herein is anL-DOPA butyl ester linked via one of the hydroxyls on the benzene to oneGABA group, and having either of the structures presented below (alsoreferred to herein as Compounds 21a and 21b):

According to some embodiments of the invention, L-DOPA is conjugated toa GABA moiety, via the carboxylic group of L-DOPA, through anoxyalkylester linker.

The term “oxyalkylester” describes an alkylene, as defined herein,linked to a carboxylic ester moiety. This term, for example, encompassesa —(CH₂)m-O—C(═O)— group, where m can be 1, 2, 3, 4, and up to 10. Insome embodiments, the oxyalkylester linker is an oxymethyl carboxylicester linker. Such a linker is susceptible to dissociation bybrain-derived enzymes and is further highly advantageous by releasing,upon its dissociation, an additional metabolite—formaldehyde, which canalso exhibit a beneficial pharmacological effect.

In some embodiments, when the oxyalkylester linker is oxymethylester,the conjugate has the following structure (also referred to herein asCompound 33):

The conjugates presented herein can be collectively represented by thegeneral formula I:

wherein:

R₁-R₃ are each independently selected from the group consisting ofhydrogen, 4-amino-butyryl and butyryl; and

R₄ is selected from the group consisting of hydrogen, alkyl,butyryloxyalkyl and 4-amino butyryloxyalkyl,

such that at least one of R₁-R₃ is a 4-aminobutyryl and/or R₄ is4-aminobutyryloxyalkyl.

Thus, at least one of R₁-R₄ represents a GABA moiety, as defined herein,formed by coupling GABA to L-DOPA.

According to some embodiments, only one of R₁-R₄ is a GABA moiety.According to some embodiments, R₁ is 4-amino-butyryl (GABA moiety) andR₃ and R₂ are each hydrogen. In these embodiments, the GABA moiety iscoupled to the amine group of L-DOPA via an amide bond.

According to some embodiments, R₂ is 4-amino-butyryl (GABA moiety) andR₁ and R₃ are each hydrogen. In these embodiments, the GABA moiety iscoupled to a hydroxyl group of L-DOPA via an ester bond.

According to some embodiments, R₃ is 4-amino-butyryl (GABA moiety) andR₁ and R₂ are each hydrogen. In these embodiments, the GABA moiety isalso coupled to a hydroxyl group of L-DOPA via an ester bond.

According to some embodiments, the conjugate comprises more than oneGABA moiety, and thus in some cases, the compound having the generalformula I, R₁ and R₂ are each a GABA moiety and R₃ is hydrogen. In somecases, the compound is having the general formula I, R₃ and R₂ are eacha GABA moiety and R₁ is hydrogen (for example, compound 21). Asdemonstrated in the Examples section that follows, such compounds arereferred to as bis-GABA conjugates.

According to other embodiments, the compound includes three GABAmoieties, and thus three of R₁-R₄ in formula I is a 4-amino-butyrylgroup (GABA moiety).

In some embodiments, each of R₁-R₃ is a 4-amino-butyryl group (GABAmoiety).

In some embodiments, the carboxylic acid group in the L-DOPA moiety isin its free acid form, namely a —(C═O)OH form, and thus R₄ in formula Iis hydrogen.

According to other embodiments, the L-DOPA moiety is in its ester form,and thus R₄ is an alkyl, such as, but not limited to, methyl, ethyl,propyl, butyl and octyl. In some embodiments, R₄ is methyl and in otherembodiments, R₄ is butyl.

As demonstrated in the Examples section that follows, the free acidform, as well as the methyl and butyl ester forms, of various conjugatesaccording to embodiments of the invention, have been prepared.

Exemplary such conjugates include Compounds 16, 21, 21a and 21b.

According to embodiments of the invention, exemplary L-DOPA-GABAconjugates include compounds encompassed by general Formula I, wherein:

R₁ is 4-amino-butyryl (GABA moiety), R₃ and R₂ are each hydrogen, and R₄is hydrogen, methyl or butyl (for example, Compound 5 (BL-1023) andCompound 16 (BL-1023*));

R₂ is 4-amino-butyryl (GABA moiety), R₁ and R₃ are each hydrogen, and R₄is hydrogen, methyl or butyl (for example, Compound 21b);

R₃ is 4-amino-butyryl (GABA moiety), R₁ and R₂ are each hydrogen, and R₄is hydrogen, methyl or butyl (for example, Compounds 21a);

R₁ and R₂ are each 4-amino-butyryl (GABA moiety), R₃ is hydrogen, and R₄is hydrogen, methyl or butyl;

R₁ and R₃ are each 4-amino-butyryl (GABA moiety), R₂ is hydrogen, and R₄is hydrogen, methyl or butyl;

R₂ and R₃ are each 4-amino-butyryl (GABA moiety), R₁ is hydrogen, and R₄is hydrogen, methyl or butyl (e.g., Compound 21 (AN-490)); and

each of R₁-R₃ is 4-amino-butyryl (GABA), and R₄ is hydrogen, methyl orbutyl.

As further demonstrated in the Examples section that follows, aconjugate in which the L-DOPA is linked via its carboxylic group to abutyryloxyalkyl moiety has also been prepared (see, for example,Compound 33).

A butyryloxyalkyl moiety represents, for example, —(CH₂)m-O—C(═O)-butyl,which is linked to the carboxylate (—C(═O)—O—) group derived from thecarboxylic acid group of L-DOPA. Accordingly, a 4-aminobutyryloxymethylgroup represents —CH₂—O—C(═O)—(CH₂)₃—NH₂.

The advantageous pharmacological features of such a conjugate aredelineated hereinabove.

In addition, while, as described in detail in the Examples section thatfollows, coupling a GABA moiety selectively to either or both hydroxylgroups of L-DOPA and/or to the amine group of L-DOPA requires syntheticand purification manipulations that may affect the overall synthesisyield, coupling a GABA moiety to the carboxylic acid group of L-DOPA isperformed selectively without excessive manipulations.

Using this form of linking GABA to the carboxylic acid moiety of L-DOPA,conjugates in which R₄ is a GABA moiety are prepared.

Thus, according to embodiments of the invention, the conjugate has thegeneral formula I hereinabove, in which R₄ is a butyryloxyalkyl, andeach of R₁-R₃ is hydrogen.

According to further embodiments, R₄ is a butyryloxyalkyl, and at leastone of R₁-R₃ is 4-aminobutyryl.

According to further embodiments, R₄ is a butyryloxyalkyl, and at leasttwo of R₁-R₃ is 4-aminobutyryl.

According to further embodiments, R₄ is a butyryloxyalkyl, and each ofR₁-R₃ is 4-aminobutyryl, such that the conjugate comprises 4 GABAmoieties linked to L-DOPA.

According to some embodiments, the conjugates presented herein andrepresented by compounds having the general formula I are is an acidaddition salt form thereof.

As is well known in the art, the phrase “acid addition salt” describes acomplex of two ionizable moieties, a base and an acid, which, wheninteracted in a particular stoichiometric proportion and under suitableconditions, form a salt that comprises one or more cations of the basemoiety and one or more anions of the acid moiety. As used herein, thephrase “acid addition salt” refers to such a complex, in which the basemoiety is an amine, such that the salt comprises a cationic form of theamine (ammonium) and an anionic form of an acid.

The amine group can be derived from the GABA moiety or moieties linkedto L-DOPA, or from the amine moiety of L-DOPA, if present in anon-conjugated form thereof.

Depending on the stoichiometric proportions between the base and theacid in the salt complex, as is detailed hereinbelow, the acid additionssalts can be either mono addition salts or poly addition salts.

The phrase “mono addition salt”, as used herein, refers to a saltcomplex in which the stoichiometric ratio between the acid anion andamine cation is 1:1, such that the acid addition salt includes one molarequivalent of the acid per one molar equivalent of the conjugate.

The phrase “poly addition salt”, as used herein, refers to a saltcomplex in which the stoichiometric ratio between the acid anion and theamine cation is greater than 1:1 and is, for example, 2:1, 3:1, 4:1 andso on, such that the acid addition salt includes two or more molarequivalents of the acid per one molar equivalent of the conjugate.

The stoichiometric proportions between the base and the acid of the saltcomplex, according to some embodiments of the present invention, rangesfrom 6:1 to 1:6 base:acid equivalents, from 4:1 to 1:4 base:acidequivalents, from 3:1 to 1:3 base:acid equivalents or from 1:1 to 1:3base:acid equivalents.

The acid addition salts of a chemical conjugate according to the presentinvention are therefore complexes formed between one or more aminogroups of the compound and one or more equivalents of an acid. The acidaddition salts may therefore include a variety of organic and inorganicacids, such as, but not limited to, hydrochloric acid which affords anhydrochloric acid addition salt, acetic acid which affords an aceticacid addition salt, ascorbic acid which affords an ascorbic acidaddition salt, benzenesulfonic acid which affords a benzenesulfonic acidaddition salt, camphorsulfonic acid which affords a camphorsulfonic acidaddition salt, toluenesulfonic acid which affords a toluenesulfonic acidaddition salt, trifluoroacetic acid which affords a trifluoroacetic acidaddition salt, citric acid which affords a citric acid addition salt,maleic acid which affords a maleic acid addition salt, methanesulfonicacid which affords a methanesulfonic acid (mesylate) addition salt,naphthalenesulfonic acid which affords a napsylate addition salt, oxalicacid which affords an oxalic acid addition salt, phosphoric acid whichaffords a phosphoric acid addition salt, succinic acid which affords asuccinic acid addition salt, sulfuric acid which affords a sulfuric acidaddition salt and tartaric acid which affords a tartaric acid additionsalt. Each of these acid addition salts can be either a mono acidaddition slat or a poly acid addition salt, as these terms are definedhereinabove.

Any other pharmaceutically acceptable salts, solvates or hydrates of theconjugates described herein, as well as processes of preparing thesepharmaceutically acceptable salts, solvates and hydrates, are alsoencompassed by embodiments of the invention.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the hybrid compound) and a solvent, whereby the solvent does notinterfere with the biological activity of the solute. Suitable solventsinclude, for example, ethanol, acetic acid and the like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

Embodiments of the invention further encompass various crystalline forms(polymorphs) of the conjugates described herein, as well as processes ofpreparing these crystalline forms.

Embodiments of the invention further encompass all isomeric forms,stereoisomeric forms (including enantiomers and diastereomers) andracemic mixtures of the conjugates described herein.

The conjugates presented herein, by being capable of effectivelycrossing the BBB and releasing free dopamine and free GABA in the braintissue, can be beneficially used in various therapeutic applications inwhich elevated levels of dopamine and GABA in the brain are desired.These include, for example, treating or preventing a neurodegenerativedisease or disorder.

Hence, according to an additional aspect of embodiments of theinvention, the conjugate described herein can be used as a medicament,whereby the medicament can be utilized in treating or preventing aneurodegenerative disease or disorder.

According to an additional aspect of embodiments of the invention thereis provided a method of treating a neurodegenerative disease ordisorder. The method, according to these embodiments, is effected byadministering to a subject in need thereof a therapeutically effectiveamount of the conjugate as presented herein.

The term “treating” used herein refers to refers to reversing,alleviating, inhibiting the progress of, or preventing the disorder,disease or condition to which such term applies, or one or more symptomsof such disorder or condition. The term “treatment” or “therapy” as usedherein refer to the act of treating.

The phrase “neurodegenerative disease or disorder” as used herein refersto any disease, disorder or condition of the nervous system (e.g., thecentral nervous system, CNS) which is characterized by gradual andprogressive loss of neural tissue, neurotransmitter, or neuralfunctions. Examples of neurodegenerative disorder include, Parkinson'sdisease, multiple sclerosis, amyatrophic lateral sclerosis, autoimmuneencephalomyelitis, Alzheimer's disease and Huntington's disease.

According to some embodiments the neurodegenerative disease isParkinson's disease.

The conjugates described herein can be administered by any acceptableroute of administration.

In some embodiments, the conjugate is administered parenterally (e.g.,subcutaneously or intraperitoneally) or orally.

The term “subject” as used herein refers to a mammal having a bloodbrain barrier, such as a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the conjugate being administered which is capable ofrelieving to some extent one or more of the symptoms of theneurodegenerative disease or disorder being treated.

A therapeutically effective amount according to some embodiments of theinvention ranges between 0.01 and 200 mg/kg body, between 0.1 and 100mg/kg body, between 0.5 and 50 mg/kg body, or between 1 and 20 mg/kgbody.

In each of the methods and used described herein, the conjugatespresented herein can be utilized in combination with an additionalactive agent.

In some embodiments, the additional active agent is a CNS-acting agent.

As used herein, the phrase “CNS-acting agent” encompasses any compoundwhich is capable of exerting a CNS activity.

The phrase “CNS activity” as used herein describes a pharmacologicalactivity exerted in the CNS, which is aimed at treating a CNS-associatedimpairment. Such a pharmacological activity typically includesmodulation of neuronal signals transduction.

According to one embodiment, the CNS-acting agent is a psychotropicdrug.

Psychotropic drugs are known in the art, and are referred to herein, aspharmacological agents that exert activity in the CNS to thereby treat aCNS-associated disease or disorder.

Psychotropic drugs include, but are not limited to, anti-psychotic drugs(typical and atypical), anxiolytic drugs, anti-depressants,anti-convulsive drugs (also referred to herein and is the art andanti-convulsants), anti-parkinsonian drugs, acetylcholine esteraseinhibitors, MAO inhibitors, selective serotonin reuptake inhibitors(SSRIs) and selective noradrenalin receptor inhibitors (SNRIs).

Representative examples of psychotropic drugs that can be utilized incombination with the conjugates of the present embodiments include,without limitation, chlorpromazine, perphenazine, fluphenazine,zuclopenthixol, a thiopropazate, haloperidol, benperidol, bromperidol,droperidol, spiperone, pimozide, piperacetazine, amilsulpride,sulpiride, clothiapine, ziprasidone, remoxipride, sultopride,alizapride, nemonapride, clozapine, olanzapine, ziprasidone, sertindole,quetiapine, fluoxetine, fluvoxamine, desipramine, paroxetine,sertraline, valproic acid, temazepam, flutemazepam, doxefazepam,oxazepam, lorazepam, lormetazepam, cinolazepam, flutazolam, lopirazepam,meprobamate, carisoprodol, acetophenazine, carphenazine, dixyrazine,priciazine, pipothiazine, homophenazine, perimetazine, perthipentyl,flupentixol, piflutixol, teflutixol, oxypethepin, trifluperidol,penfluridol, meclobemide, norclomipramine, amoxapine, nortriptyline,protriptyline, reboxetine, tacrine, rasagiline, amantidine, duloxetine,phenobarbital, phenytoin, a drug of the phenothiazines family, a drug ofthe benzodiazepines family and butyrophenone.

The conjugates presented herein can be utilized in any of the uses andmethods described herein either per se or as part (as an activeingredient) of a pharmaceutical composition.

As used herein a “pharmaceutical composition” refers to a preparationthat comprises the conjugate as described herein and other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Pharmaceuticalcompositions of the present invention may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with embodiments ofthe invention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the conjugates presented herein may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol. For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the conjugates presented herein can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the conjugates of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the conjugate may take the form of tablets orlozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according toembodiments of the invention are conveniently delivered in the form ofan aerosol spray presentation from a pressurized pack or a nebulizerwith the use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The conjugates described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active compound in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theconjugates to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The conjugates presented herein may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of embodimentsof the invention include the active ingredients contained in an amounteffective to achieve the intended purpose. More specifically, atherapeutically effective amount means an amount of conjugate effectiveto prevent, alleviate or ameliorate symptoms of a neurodegenerativedisease or disorder.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For the conjugate used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in cell cultures and/or animals. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that includes the IC50 as determined by activity assays (e.g., theconcentration of the test compound, which achieves a half-maximalactivity in the dopamine and GABA systems). Such information can be usedto more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the conjugate described herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the IC50 and the LD50 (lethal dose causingdeath in 50% of the tested animals) for a subject compound. The dataobtained from these activity assays and animal studies can be used informulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active ingredients which are sufficient to maintainclinically beneficial effects, termed the minimal effectiveconcentration (MEC). Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a controlledreleased formulation.

The phrase “controlled release” as used herein refers to a formulationcapable of releasing the active ingredient at a predetermined rate suchthat therapeutically beneficial levels are kept over an extended periodof time. Suitable controlled release formulations are well known in theart (e.g., “Remington's Pharmaceutical Sciences,” Philadelphia Collegeof Pharmacy and Science, 19th Edition, 1995).

The amount of a pharmaceutical composition to be administered will, ofcourse, be dependent on the subject being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc.

The pharmaceutical composition of the present invention may, if desired,be presented in a pack or dispenser device, such as a FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccompanied by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Pharmaceutical compositions comprising theconjugate presented, optionally in combination with an additional activeagent, formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as set forth hereinabove.

Thus, according to an embodiment of this aspect of the presentinvention, the pharmaceutical composition is packaged in a packagingmaterial and identified in print, in or on the packaging material, foruse in the treatment of a neurodegenerative disease or disorder asdescribed herein.

According to some embodiments, the pharmaceutical composition ispackaged in a packaging material and identified in print, in or on thepackaging material, for use in the treatment of a Parkinson's disease.

Such a packaged pharmaceutical composition is also referred to hereininterchangeably as an article-of-manufacturing or a pharmaceutical kit.

Hence, the invention provides novel conjugates, pharmaceuticalcompositions, articles-of-manufacturing and methods of use thereof fortreating neurodegenerative disorders safely and effectively.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non limiting fashion.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Chemical Syntheses of L-DOPA-GABA Conjugates

Materials and Methods:

¹H and ¹³C-NMR spectra were obtained on Bruker AC-200, DPX-300 andDMX-600 spectrometers. Chemical shifts are expressed in ppm downfieldfrom Me₄Si (TMS) used as internal standard. The values are given in δscale.

HRMS/LRMS were obtained on an AutoSpec Premier (Waters UK) spectrometerin CI (=Chemical Ionization), CH₄.

Progress of the reactions was monitored by TLC on silica gel (Merck,Art. 5554).

Flash chromatography was carried out on silica gel (Merck, Art. 9385).Compounds with nitrogen were colored by ninhydrin.

Melting points were determined on a Fisher-Johns apparatus and wereuncorrected.

HPLC measurements were conducted using C-128 (internal number)Phenomenex Nucleosil C18 column, 5 μm, 250×4.6 mm, 120 A. An exemplarymobile phase included A: 0.1% TFA in H₂O, B: CH₃CN, and analysis wasconducted using the following gradient: 0 to 4 minutes, 0% of B; 10minutes, 30% of B; 11 minutes, 30% of B; 12 minutes, 0% of B; 14minutes, 0% of B. Flow rate: 1 ml/ml. Product was dissolved in water (1mg/ml). UV detector was operated at 282 nm. Running time: 22 minutes.Temperature: 5° C.

IR measurements were conducted using Bio-Rad FTS 3000MXspectrophotometer, for KBr pellets containing the tested compound.

Commercially available compounds were used without further purification.

Water content measurements were performed using Coulometric Karl-Fischerwith Metrohm Thermoprep 832 and Metrohm Coulometer 831 according to USP<921> method 1c.

The nomenclature of the compounds was given according to ChemDraw Ultrav. 11.0.1 (CambridgeSoft). The numbering on the chemical structures isfor spectral analysis only.

The following compounds were prepared according to known procedures:chloromethyl chlorosulfate [Binderup et al. Synth. Commun. 1984, 14,857-864], N-Boc-GABA [Bodanszky et al. Principles of Peptide Synthesis.In Springer-Verlag, New York: 1984; p 99] and N-Cbz-GABA [Lever Jr, O.W. and Vestal, B. R. J. Heterocycl. Chem. 1986, 23, 901-904].

Preparation of Mono-GABA-L-DOPA Conjugate (Compound 5, BL-1023; Having aGABA Moiety Linked to L-DOPA via an Amide Bond)

Synthetic Route 1:

The L-DOPA-GABA conjugate (BL-1023) is prepared by reacting L-DOPA andGABA and isolating the obtained conjugate. According to some embodimentspresented herein, the amine group of the GABA is protected prior to theconjugation reaction. Since L-DOPA includes a few functional groups thatcan react with the functional carboxylic acid group of GABA, typically,a conjugate that includes L-DOPA and at least two GABA molecules isobtained, requiring an additional step of removing a

GABA molecule selectively so as to obtain the desired product.Thereafter, removal of the amine-protecting group in the GABA moiety iseffected.

As detailed hereinbelow, using this pathway, the L-DOPA-GABA conjugatehaving a single GABA moiety (mono-GABA-L-DOPA conjugate) wassuccessfully obtained in high yield and purity, upon carefully selectingthe reaction conditions. The synthetic pathway for preparing L-DOPA-GABAconjugate (Compound 5, BL-1023) is illustrated in

Scheme 1 below.

Preparation of N-Protected GABA, Boc-N-4-aminobutyric Acid (N-ProtectedBoc-GABA, Compound 1)

A 6-Liter three-necked round-bottom flask equipped with a cooling bathand a thermometer was charged with 167 grams (1.62 mol) of4-aminobutanoic acid in 1 Liter dioxane and 0.5 Liter water. 450.5 ml(3.24 mol) triethylamine was added and the mixture was stirred for 20minutes and cooled to 10° C. 353.2 grams (1.62 mol) Boc anhydride wasthen added at 15° C. and the mixture was stirred for 12 hours at 17° C.5 Liters 0.5N HCl was thereafter added to the reaction mixture, theproduct was extracted with 5 Liters ethyl acetate and the extract waswashed with 3×5 Liters brine and dried over MgSO₄ overnight. The solventwas evaporated under reduced pressure to afford 300 grams (91% yield) ofCompound 1 (see, Scheme 1). The product structure was confirmed by¹H-NMR.

Preparation of L-DOPA Methyl Ester (Compound 2):

A 3-Liter three-necked round-bottom flask equipped with a cooling bath,a thermometer and a condenser was charged with 1.5 Liters methanol, andacetyl chloride (119 ml, 1.2 mol) was carefully added thereto at 0° C.The cooling bath was replaced with a heating mantle and the mixture washeated to 15° C. 60 grams (0.3 mol) of L-3-(3,4-dihydroxy-phenyl)alaninewas then added and the reaction mixture was refluxed for 3 hours andthereafter concentrated under reduced pressure at 60° C. to afford 74.3grams (100% yield) of Compound 2 (see, Scheme 1). The product structurewas confirmed by ¹H-NMR.

Preparation of di-GABA-L-DOPA Conjugates (Compounds 3A and 3B):

A 4-Liter round-bottom flask equipped with a nitrogen inlet, motorstirrer, additional funnel and thermometer was charged with 114.8 grams(0.56 mol) Compound 1 in 2 Liters dichloromethane (DCM) and 90 gramscarbonyl diimidazole (CDI, 0.56 mol) were added portion wise thereto at17° C. with stirring. The mixture was stirred for 3 hours and 70 grams(0.28 mol) of Compound 2, followed by 48.4 grams N-ethyldiisopropylamine(0.28 mol) were added. The reaction mixture was stirred for 24 hours at17° C., washed in a separation funnel with 2×1.5 Liters 0.1N HCl and 3×1Liter water and the organic phase was dried over MgSO₄ overnight. Thesolvent was remover under reduced pressure and the residue was purifiedby column chromatography using a silica gel column and a 1:2 mixture ofethyl acetate:hexane mixture as eluent to afford 120 grams (73% yield)of a mixture of Compounds 3A and 3B (see, Scheme 1). The presence of amixture of these two isomers was confirmed by ¹H-NMR.

Preparation of Boc-N-GABA-L-DOPA Conjugate (Compound 4):

A 3-Liter three-necked round-bottom flask equipped with a nitrogeninlet, a thermometer and a heating mantle was charged with 120 grams(0.2 mol) of a mixture of Compounds 3A and 3B dissolved in 500 mlmethanol. 24 grams NaOH (0.6 mol) were then added and the reactionmixture was heated at 60° C. for 1 hour. The reaction mixture was thenconcentrated under reduced pressure, 650 ml 1N HCl were added at 5° C.and the product was extracted with ethyl acetate. The extract was washedwith brine, dried over MgSO₄ overnight and the solvent was remover underreduced pressure. The residue was purified by column chromatography (soas remove traces of Compound 1) using a silica gel column, under argonatmosphere, and a 1:2 mixture of 0-5% methanol in DCM as eluent, toafford 25 grams (32% yield) of Compound 4 (see, Scheme 1). The productstructure was confirmed by ¹H-NMR.

Preparation of L-DOPA-GABA Conjugate (Compound 5, BL-1023):

A 500 ml three-necked round-bottom flask equipped with a nitrogen inlet,a thermometer and a cooling bath was charged with 25 grams (0.065 mol)of Compound 4 dissolved in 200 ml ethyl acetate. The solution was cooledto 0° C. and 49 ml (0.196 mol) 4N HCl in dioxane were added thereto andmixture was stirred at 17° C. for 24 hours. The solid product wasfiltered and washed with 4×200 ml ethyl acetate and dried at 60° C., 10mmHg, for 9 hours to afford 15 grams (72%) of Compound 5 (see Scheme 1and Compound I hereinabove). The product structure was confirmed by¹H-NMR but significant amounts of dioxane and ethyl acetate wereobserved.

Removal of the solvents was performed by dissolving 37 grams of Compound5 (obtained in 3 batches according to the procedure describedhereinabove) in 150 ml isopropanol (IPA) and pouring the solution to 3Liters diethyl ether while vigorously stirring the resulting mixture.The solid product was filtered, washed thoroughly with 3×200 ml etherand dried at 100° C., 10 mmHg, for 60 hours to afford Compound 5 as abrown powder. ¹H-NMR indicated the presence of 0.2% ether.

Purity of the product was determined by HPLC to be 99.5%.

Water content was determined by Karl-Fisher analysis to be 0.41%.

Elemental analysis: C:47.67%; H:6.24%; N:8.55%.

Chloride analysis: 9.62%.

GC-MS analysis showed data consistent with molecular mass (283.8 forM-HCl).

¹³C-NMR: δ=22.55, 31.82, 38.43, 53.81, 116.1 (d), 121.28, 128.97, 142.85(d), 174.39 (d) ppm.

Synthetic Route 2 (Up-Scaled):

The following presents an improved, scaled-up synthesis protocol forobtaining Compound 5, as schematically described in Scheme 2 below.

Preparation of L-DOPA methyl ester (Compound 2): A 5-Liter three-neckedround-bottom flask equipped with a cooling bath, a thermometer and acondenser was charged with methanol (2.5 Liters). Acetyl chloride (2.03mol, 4 equivalents) was added carefully at 0° C., the reaction mixturewas thereafter cooled to room temperature (15° C.) and L-DOPA (0.507mol, 1 equivalent) was added. The reaction mixture was refluxed (65° C.)for 3 hours and then concentrated under vacuum to afford 126.6 grams ofcompound 2 as a white solid (99% yield).

¹NMR (400 MHz, DMSO-d): δ=2.95 (m, 2H, H-2), 3.69 (s, 3H, OMe), 4.13 (s,1H, H-1), 6.45 (dd, J=1.9 Hz, J′=8 Hz, 1H, Arom. H), 6.59 (d, J=1.9 Hz,1H, Arom. H), 6.68 (d, J=8 Hz, Arom. H), 8.51 (s, 3H, NH3+), 8.87 (br,2H, OH).

Preparation of di-GABA-L-DOPA Conjugates (Compounds 3A and 3B):

Compounds 3A and 3B were obtained by reacting methylated L-DOPA salt(Compound 2) and Boc-protected GABA (Compound 1). A 3-Liter three-neckedround-bottom flask, equipped with a thermometer and being under argonatmosphere, was charged with Boc-protected GABA (0.848 mol, 2equivalents) and dichloromethane (1.5 Liter).

Carbonyldiimidazole (CDI) (0.848 mol, 2 equivalents) was added portionwise at room temperature (17° C.) with stirring, and the reactionmixture was stirred at room temperature for 3 hours. Then, Compound 2(0.424 mol, 1 equivalent) and N-ethyldiisopropylamine (0.432 mol, 1.02equivalent) were added, and the reaction mixture was stirred for 24hours at room temperature. The reaction mixture was thereafter washedwith a 0.1 M HCl solution (2×1 Liter) (in a separation funnel) and withwater (3×500 ml). The organic phase was dried over MgSO4 overnight,filtered and evaporated to afford 225.6 grams of a mixture of Compounds3A and 3B as a slightly yellow foam (91% yield).

¹H NMR spectra confirmed the presence of a mixture of the tworegioisomers Compounds 3A and 3B and of residual Boc-protected GABA(Compound 1).

Preparation of Boc-N-GABA-L-DOPA Conjugate (Compound 4):

This step involves deprotection of the methyl ester and of the phenolprotecting groups. A 2-Liter three-necked round-bottom flask equippedwith a thermometer, under argon atmosphere, was charged with a mixtureCompounds 3A and 3B (0.388 mol, 1 equivalent), obtained as describedhereinabove, in methanol (990 ml). A solution of NaOH (1.16 mol, 3equivalents) in water (1.2 Liter) was then added and the reactionmixture was heated at 60° C. for 1 hour. The reaction mixture wasthereafter concentrated in vacuum and HCl 1M (1.2 Liter) was added at 5°C. The aqueous phase was extracted with ethyl acetate (2×800 ml), theorganic layers were combined and washed with saturated NaCl (2×700 ml),dried overnight over MgSO₄, filtered and concentrated, to give a mixtureof the desired Compound 4 (approximately 180 grams) and of Boc-protectedGABA.

Compound 4 was purified by re-crystallization or precipitation. In anexemplary purification procedure, the mixture of Compound 4 andBoc-protected GABA was dissolved in ethyl acetate (200 ml) and theobtained solution was added slowly to cold dichloromethane (2 Liters)under stirring. The desired product precipitated and the solution wasdecanted. Upon 3-5 repetitive precipitations, Pure Compound 4 (70.44grams) was obtained as a slightly brown foam (47% yield).

¹H NMR (400 MHz, DMSO-d): δ=1.37 (s, 9H, H-Boc), 1.52 (m, 2H, CH2), 2.05(m, 2H, CH2), 2.75 (m, 4H, CH2), 4.29 (m, 1H, H-2), 6.45 (dd, J=1.8 Hzand J′=8 Hz, 1H, Arom. H), 6.60 (d, J=8 Hz, 2H, Arom. H), 6.77 (t, J=4.9Hz, 1H, NHBoc), 8.03 (d, J=8 Hz, 1H, NH), 8.70 (dbr, 2H, OH), 12.60 (br,1H, CO2H).

Preparation of L-DOPA-GABA Conjugate (Compound 5, BL-1023):

The final step of the synthesis involves deprotection of the aminofunction and the formation of the BL-1023 hydrochloric salt. A 3-Literthree-necked round-bottom flask equipped with a thermometer, under argonatmosphere, was charged with Compound 4 (0.184 mol, 1 equivalent) inethyl acetate (950 ml). The solution was cooled to 0° C. and HCl 2M indiethyl ether (0.553 mol, 3 equivalents) was added. The reaction mixturewas stirred at room temperature for 36 hours. The resulting precipitatewas filtered and washed with ethyl acetate (3×200 ml). The product wasthereafter dissolved in isopropanol (250 ml) and the solution was addedslowly to diethyl ether (4 Liters) under vigorous stirring. The obtainedprecipitate was filtered under inert atmosphere (Argon) and dried undervacuum, to afford the desired Compound 5 (BL-1023) (slightly beigesolid). After several days under vacuum the compound was still not dry(36 grams) and contained isopropanol, diethyl ether, ethyl acetate andwater.

¹H NMR (400 MHz, DMSO-d): δ=1.74 (m, 2H, CH2), 2.20 (m, 2H, CH2), 2.86(m, 4H, CH₂), 4.29 (m, 1H, H-2), 6.47 (dd, J=1.8 Hz and J′=8 Hz, 1H,Arom. H), 6.64 (d, J=8 Hz, 2H, Arom. H), 7.99 (br, 3H, NH₃ ⁺), 8.20 (d,J=7.9 Hz, 1H, NH), 8.77 (br, 2H, OH), 12.65 (br, 1H, CO₂H).

The NMR spectrum showed also some small impurities at 1.05-1.15 ppm, 4.8ppm and 8.3 ppm in all three batches, thus indicating thatre-crystallization should be performed.

HPLC measurements indicated a purity of 92.50%.

Purification of Compound 5 (BL-1023):

Purification was performed by dissolving Compound 5 in isopropanol andadding the obtained solution to dichloromethane (DCM, 10 times volume).The precipitation of the compound occurred slowly and the precipitatewas thereafter filtered. This procedure was conducted under strict inertatmosphere.

The purification was conducted three times as far as after twosuccessive batches the purity had significantly increased.

The three purified batches of Compound 5 were poured together, dissolvedin methanol (150-200 ml) and concentrated in vacuum in order to obtainone homogeneous batch. This procedure was repeated six times in order toremove residual isopropanol (from the purification step). The obtainedresidue was then dried for six days at 65° C. in a vacuum drying oven,so as to afford 27.8 grams of Compound 5 as a slightly brown solid.

Overall yield was 10%.

¹H NMR (400 MHz, DMSO, TMS): δ=12.66 (br, COOH, 1H), 8.73 (br, OH, 2H),8.17 (d, 3H, J=7.8 Hz, NH, 1H), 7.89 (br, 3H), 6.65-6.42 (m, 3H, Arom.H), 4.29 (m, H-2, 1H), 2.88-2.63 (m, CH₂, 4H), 2.18 (m, CH₂, 2H), 1.72(m, CH₂, 2H).

IR: υ (cm⁻¹)=3235 (s), 3075 (s), 2929 (m), 1731 (m), 1646 (s), 1525 (m),1445 (w), 1358 (w), 1287 (m), 1229 (m), 1203 (m), 1155 (w), 1115 (m),979 (w), 875 (vw), 816 (m), 792 (w), 654 (w), 616 (w), 590 (w).

Heavy Metals: <0.002%.

ROI: 1.27%.

Water content: 1.24%.

Elemental analysis: Determined: 48.02% C, 6.08% H, 8.63% N; Calculated:48.98% C, 6.01% H, 8.79% N

MS (positive mode, single quadrupol 50V): m/z=283.2

DSC (heating rate 10° C./minute): 25-250° C.; endotherm at 77-78° C.;ca. 240° C. decomposition.

Purity (HPLC): 90.12%.

Stability Studies:

One sample of Compound 5 (BL-1023) was placed in an open vial andanother sample was placed in a sealed vial and then placed on the benchtop. KF and HPLC analyses were performed after 1, 3, 4 and 7 days andthe data is presented in Table 2 below.

TABLE 2 Starting point 1 day 3 days 4 days 7 days kept sealed HPLC:99.4% KF: 0.58% HPLC: 98.7% vial on KF: 0.63% (G-25-5/06) KF: 0.41%Bench top (G-25-1/06) (G-27-1/06) slightly brownish powder Kept in aHPLC: 99.4% HPLC: 98.9% HPLC: 99.4% HPLC: 99.4% open vial KF: 0.63% KF:3.8% KF: 5% KF: 5% on bench (G-25-1/06) (G-25-2/06) (G-25-3/06)(G-25-4/06) top slightly brownish slightly brownish slightly brownishBecame one powder powder with some solid; Becoming solid piecestickiness to it one solid piece ↓ HPLC: 99.0% divided to 2 KF: 5.9%parts: (G-27-2/06) Solid stuff one solid (one solid piece piece) Brokenup HPLC: 99.0% (powder) Sticky semi- powder There's not enough materialto make KF

Preparation of Methyl Esters and Acid Addition Salts of theMono-GABA-L-DOPA Conjugate Having a GABA Moiety Linked to L-DOPA via anAmide Bond:

Methyl esters and salts of mono-GABA-L-DOPA conjugates were successfullyobtained using the synthetic pathway depicted in Scheme 3 below.

The ester Compound 6 was prepared using thionyl chloride (SOCl₂) inmethanol (MeOH) as illustrated in Scheme 3. Compound 6 was reacted withCompound 1 (Boc-GABA), prepared as described hereinabove (see,

Scheme 1). Compound 7 was synthesized by coupling Compound 6 withCompound 1 using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDCl) and N-hydroxybenzotriazole (HOBt) indimethylformamide (DMF) (see, Scheme 3). The subsequent removal of theBoc protective group was carried out under acidic conditions of 4N HClin EtOAc or with TFA to afford Compound 8a and Compound 8b.

Specifically, EDCl (1.1 equivalents) and HOBt (1.1 equivalents) wereadded to a stirred and cooled solution of Compound 1 (Boc-GABA, 1equivalent) in DMF (10 ml per mmol) in an ice bath. After 1 hour ofstirring the ice bath was removed and Compound 6 (L-DOPA ester, 1equivalent) and Et₃N (3 equivalents) were added and the pH of thereaction mixture was about 9. The mixture was stirred over night,diluted with EtOAc and washed three times with 1 M KHSO₄, three timeswith 1 M NaHCO₃, and three times with brine, dried over Na₂SO₄ andevaporated under reduced pressure to afford the crude productmethyl-2-(tert-butyl-3-carbamoylpropylcarbamate)-3-(3,4-dihydroxyphenyl)propanoate(Compound 7) as a sticky white solid (50% yield), and used in the nextsteps without further purification.

¹H-NMR (300 MHz, CDCl₃) ppm: δ=6.75 (d, J=8.4 Hz, 1H, H-5), 6.69 (bs,1H, H-2), 6.47 (dd, J=8.4, 2.1 Hz, 1H, H-6), 4.99 (bs, 1H, H-11 orH-12), 4.78 (m, 1H, H-8), 3.71 (s, 3H, H-10), 3.08-3.02 (m, 3H,H-17+H-7), 2.91 (m, 1H, H-7), 2.2 (m, 2H, H-15), 1.75 (m, 2H, H-16),1.43 (s, 9H, H-21);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=173.04 (1C, C-14), 172.35 (1C, C-9),156.99 (1C, C-19), 144.26 (1C, C-3), 143.79 (1C, C-4), 127.82 (1C, C-1),121.05 (1C, C-6), 116.38 (1C, C-5), 115.36 (1C, C-2), 80.13 (1C, C-20),53.75 (1C, C-8), 52.55 (1C, C-10), 39.91 (1C, C-17), 37.12 (1C, C-7),33.48 (1C, C-15), 28.51 (3C, C-21), 21.16 (1C, C-16);

MS analysis (TOF, ES⁺) m/z for C₁₉H₂₈N₂O₇ (calculated=396.19):[MH⁺]=397; [M+Na⁺]=419; [MH⁺-Boc]=297.

N-Boc deprotection solution was prepared by slowly adding acetylchloride (28.5 ml) to an ice-cold solution of EtOAc (20 ml) and EtOH(23.5 ml) in a 250 ml flame dried flask, equipped with a drying tube andmagnetic stirrer, followed by the addition of EtOAc to a total volume of100 ml. The ice bath was thereafter removed and the solution was usedimmediately.

The N-Boc protecting group was removed by dissolving Compound 7 in afreshly prepared solution of 4N HCl in EtOAc. The reaction mixture wasstirred for 1 hour at room temperature, and thereafter the solvent wasevaporated under reduced pressure to afford the crude deprotectedproduct methyl-2-(4-aminobutanamide)-3-(3,4-dihydroxyphenyl)propanoatehydrochloride (Compound 8a) was obtained as a white solid (81% yield).

¹H-NMR (300 MHz, D₂O) ppm: δ=6.83 (d, J=7.85 Hz, 1H, H-5), 6.74 (bs, 1H,H-2), 6.66 (bd, J=7.85 Hz, 1H, H-6), 4.64 (m, 1H, H-8), 3.72 (s, 3H,H-10), 3.09 (m, 1H, H-7), 2.82 (m, 3H, H-7+H-17), 2.31 (td, J=7.5, 2.4Hz, 2H, H-5), 1.81 (quint, J=7.5 Hz, 2H, H-16);

¹³C-NMR (200 MHz, D₂O) ppm: δ=176.59 (1C, C-14), 175.72 (1C, C-9),145.89 (1C, C-3 or C:4), 144.87 (1C, C-3 or C-4), 131.22 (1C, C-1),123.56 (1C, C-6), 118.88 (1C, C-2 or C-5), 118.25 (1C, C-2 or C-5),56.13 (1C, C-8), 54.92 (1C, C-10), 40.66 (1C, C-17), 37.93 (1C, C-7),34.05 (1C, C-15), 24.81 (1C, C-16);

HRMS analysis (CH₄) m/z for C₁₄H₂₀N₂O₅ (calculated=296.137):[M]=296.147; [MH⁺]=297.146; [MH⁺-NH₃]=280.124;

Chemical analysis calculated for C₁₄H₂₁ClN₂O₅.0.5H₂O: C 49.23%, H 6.90%,N 8.04% and O 25.67%.

Compound 7 (370 mg, 0.93 mmol) was dissolved in trifluoroacetic acid(TFA, 10 ml) and stirred at room temperature for 1 hour and TFA wasevaporated under reduced pressure. The oily residue was dissolved inether and evaporated under reduced pressure to give the product methyl2-(4-aminobutanamide)-3-(3,4-dihydroxyphenyl)propanoate2,2,2-trifluoroacetatesalt (Compound 8b) as a sticky white solid in quantitative yield.

¹H-NMR (200 MHz, D₂O) ppm: δ=6.82 (d, J=7.1 Hz, 1H, H-5), 6.72 (bs, 1H,H-2), 6.64 (dd, J=7.1, 2.4 Hz, 1H, H-6), 4.64 (m, 1H, H-8), 3.05 (dd,J=14.3, 4.8 Hz, 1H, H-7), 2.82 (m, 3H, H-7+H-15), 2.29 (t, J=7.1 Hz, 2H,H-13), 1.79 (quint, J=7.1 Hz, 2H, H-14);

¹³C-NMR (200 MHz, D₂O) ppm: δ=176.54 (1C, C-12), 175.72 (1C, C-9),164.34 (1C, C-17), 145.86 (1C, C-3 or C-4), 144.83 (1C, C-3 or C-4),131.28 (1C, C-1), 123.56 (1C, C-6), 118.89 (1C, C-2 or C-5), 118.23 (1C,C-2 or C-5), 56.02 (1C, C-8), 50.90 (1C, C-10), 40.63 (1C, C-15), 37.91(1C, C-7), 34.04 (1C, C-13), 24.78 (1C, C-14);

HRMS analysis (DCI, CH₄) m/z for C₁₄H₂₀N₂O₅ (calculated=296.137):[MH⁺]=297.145.

Preparation of bis-GABA-L-DOPA Methyl Ester (Having Two GABA MoietiesLinked to L-DOPA via Carboxylic Ester Bonds)

Compounds having two GABA moieties conjugated to L-DOPA weresuccessfully prepared as illustrated in Scheme 4 below. The diesterCompound 11 was prepared using 2 equivalents of Compound 1 (Boc-GABA)and Boc-protected-L-DOPA ester (Compound 9, Scheme 4), which affordedCompound 10 at a 80% yield, followed by N-deprotection thereof to affordCompound 11 at 61% yield.

A solution of 1M NaOH (1 equivalent) was added to a solution Compound 6(L-DOPA ester, 1 equivalent) in dioxane (2 ml per mmol) and water (1 mlper mmol). After 10 minutes Boc₂O (1 equivalent) was added and thereaction mixture was stirred at room temperature over night. Thereafterthe reaction mixture was concentrated under reduced pressure and theresulting residue was dissolved in EtOAc, washed three times with 1MKHSO₄, three times 1M NaHCO₃, three times brine, and dried over Na₂SO₄and evaporated under reduced pressure to afford the crude product,tert-butyl-1-(methoxycarbonyl)-2-(3,4-dihydroxyphenyl)ethylcarbamate(Compound 9).

Compound 9 was obtained at 94% yield and used for the next step withoutfurther purification.

¹H-NMR (300 MHz, CDCl₃) ppm: δ=6.73 (d, J=7.99 Hz, H-5), 6.64 (bs, 1H,H-2), 6.49 (dd, J=7.99, 1.8 Hz, 1H, H-6), 5.10 (d, J=8.1 Hz, 1H), 4.49(m, 1H, H-8), 3.71 (s, 3H, H-10), 2.93 (m, 2H, H-7), 1.40 (s, 9H, H-16);

¹³C-NMR (200 MHz, CDCl₃) ppm: δ=172.69 (1C, C-9), 155.55 (1C, C-14),143.97 (1C, C-3 or C-4), 143.13 (1C, C-3 or C-4), 128.26 (1C, C-1),121.47 (1C, C-6), 116.17 (1C, C-2 or C-5), 115.34 (1C, C-2 or C-5),80.45 (1C, C-15), 54.67 (1C, C-8), 52.31 (1C, C-10), 37.64 (1C, C-7),28.26 (3C, C-16);

MS analysis (TOF, ES⁺) m/z for C₁₅H₂₁NO₆ (calculated=311.14):[M+Na⁺]=334.

EDCl (2.2 equivalents) and HOBt (2.2 equivalents) were added to anice-cold and stirred solution of Compound 1 (Boc-GABA, 2 equivalents) inDMF (10 ml per mmol). After 1 hour the ice bath was removed and Compound9 (N-Boc-L-DOPA methyl ester, 1 equivalent) and Et₃N (6 equivalents)were added thereto, raising the pH to higher than 10. The reactionmixture was stirred over night, diluted with EtOAc and washed with 1MKHSO₄ (three times), 1M NaHCO₃ (three times), brine (three times), driedover Na₂SO₄ and evaporated under reduced pressure to afford the crudeproducttert-butyl-1-(methoxycarbonyl)-2-(3,4-[di-tert-butyl-4-butoxyphenylcarbamate])-dihydroxyphenyl)ethylcarbamate(Compound 10).

Compound 10 was obtained as a white solid at 81% yield, dissolved inDMF, and used in the next step without further purification.

¹H-NMR (300 MHz, CDCl₃) ppm: δ=7.11 (d, J=8.14 Hz, 1H, H-5), 7.00 (dd,J=8.14, 1.25 Hz, 1H, H-6), 6.97 (d, J=1.25 Hz, 111, H-2), 4.55 (m, 1H,H-8), 3.69 (s, 3H, H-10), 3.21 (m, 4H, H-18+H-26), 3.06 (t, J=4.38 Hz,2H, H-7), 2.57 (t, J=7.30 Hz, 4H, H-16+H-24), 1.89 (m, 4H, H-17+H-25),1.43-1.41 (two overlapping singlets, 29H, H-14+H-22+H-30);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=172.13 (2C, C-15+C-23), 170.58 (1C,C-9), 156.20 (3C, C-12+C-20+C-28), 141.90 (1C, C-3), 141.03 (1C, C-4),135.05 (1C, C-1), 127.43 (1C, C-6), 124.39 (1C, C-5), 123.49 (1C, C-2),80.19+79.49 (3C, C-13+C-21+C-29), 54.36 (1C, C-8), 52.48 (1C, C-10),39.91 (2C, C-18+C-26), 37.79 (1C, C-7), 31.39 (2C, C-16+C-24), 28.53(6C, C-22+C-30), 28.39 (3C, C-14), 25.43 (2C, C-17+C-25);

MS analysis (TOF, ES⁺) m/z for C₃₃H₅₁N₃O₁₂: (calculated=681.35):[M+Na⁺]=704.

N-Boc deprotection of Compound 10 was performed by dissolving Compound10 in a freshly prepared solution of 4N HCl in EtOAc, prepared asdescribed hereinabove. The mixture was stirred for 1 hour at roomtemperature, and the solvent was evaporated under reduced pressure toafford the crude productmethyl-2-amino-3-(3,4-phenyl-di-[4-aminobutanoate])propanoate-trihydrochloride(Compound 11).

Crude Compound 11 was washed with ether to afford a yellowish solid thatwas recrystallized from MeOH/ether to afford Compound 11 at 82% yield.

Melting point (mp): 185° C.;

¹H-NMR (300 MHz, D₂O) ppm: δ=7.32+7.31 (two overlapping picks, 2H,H-2+H-5), 7.25 (bs, 1H, H-6), 4.47 (t, J=6.78 Hz, 1H, H-8), 3.84 (s, 3H,H-10), 3.41-3.25 (m, 2H, H-7), 3.12 (t, J=7.91 Hz, 4H, H-15+H-20), 2.82(t, J=7 Hz, 4H, H-13+H-18), 2.07 (m, 4H, H-14+H-19);

¹³C-NMR (200 MHz, D₂O) ppm: δ=174.75 (2C, C-12+C-17), 171.77 (1C, C-9),143.64 (1C, C-3 or C-4), 143.09 (1C, C-3 or C-9), 135.85 (1C, C-1),130.71 (1C, C-6), 126.63 (1C, C-2 or C-5), 126.32 (1C, C-2 or C-5),55.94 (1C, C-8 or C-10), 55.78 (1C, C-8 or C-10), 40.70 (2C, C-15+C-20),36.94 (1C, C-7), 32.43 (2C, C-13+C-18), 24.01 (2C, C-14+C-19);

HRMS analysis (DCI, CH₄) m/z for C₁₈H₂₇N₃O₆ (calculated=381.190:[M]=381.190;

Chemical analysis for C₁₈H₂₇N₃O₆.H₂O: C 42.70%; H 6.56%; N 8.67%; O22.31%.

Preparation of tris-GABA-L-DOPA Methyl Ester (Having Three GABA MoietiesLinked to L-DOPA via Ester and Amide Bonds)

The tris-GABA-L-DOPA (Compound 13) was prepared using 3 equivalents ofCompound 1 (N-Boc-GABA) and Compound 6, followed by deprotection of theresulting Compound 12, at 83% yield (Scheme 5).

2-[(N-4-Aminobutyramido)-3-(3,4-bis-(4-tert-butoxycarbonyl-aminobutyryloxy)phenyl]propionate methyl ester (Compound 12) was obtained asa white solid in 84% yield and used without further purification.

¹H NMR (300 MHz, CDCl₃) ppm: δ=7.07 (d, J=7.53 Hz, 1H, H-5), 6.99 (d,J=7.53 Hz, 1H, H-6), 6.66 (bs, 1H, H-2), 4.81 (m, 1H, H-8), 3.69 (s, 3H,H-10), 3.21-2.99 (m, 8H, H-7+H-15+H-23+H-31), 2.55 (t, J=7.47 Hz, 4H,H-21+H-29), 2.19 (t, J=7.47 Hz, 2H, H-13), 1.87 (m, 4H, H-22+H-30), 1.71(m, 2H, H-14), 1.40 (bs, 27H, H-19+H-27+H-35);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=172.71 (1C, C-12), 171.94+170.72 (3C,C-9+C-20+C-28), 156.53+156.23 (3C, C-17+C-25+C-33), 141.81 (1C, C-3),140.95 (1C, C-4), 135.00 (1C, C-1), 127.23 (1C, C-6), 124.54 (1C, C-5),123.47 (1C, C-2), 79.37 (3C, C-18+C-26+C-39), 53.09 (1C, C-8), 52.54(1C, C-10), 39.88 (2C, C-23+C-31), 37.07 (1C, C-15), 33.33 (1C, C-13),31.31 (2C, C-21+C-29), 28.49 (9C, C-19+C-27+C-35), 26.13 (1C, C-14),25.37 (2C, C-22+C-30);

MS analysis (TOF, ES⁺) m/z for C₃₇H₅₈N₄O₁₃ (calculated=766.40):[MH⁺]=767; [M+Na⁺]=789.

2-[(N-4-Aminobutyramido)-3-(3,4-bis-(4-aminobutyroyloxy)phenyl]propanoate methyl ester tri-hydrochloride (Compound13) was obtained from Compound 12 as a white powder in quantitativeyield.

Melting point (mp): 87° C.;

¹H-NMR (300 MHz, D₂O) ppm: γ=7.26 (d, J=1.2 Hz, 2H, H-5+H-6), 7.19 (bt,J=1.2 Hz, 1H, H-2), 4.73 (m, 1H, H-8), 3.75 (s, 3H, H-10), 3.28 (dd,J=14, 5.8 Hz, 1H, H-7), 3.11 (bt, J=7.5 Hz, 4H, H-19+H-23), 3.03 (m, 1H,H-7), 2.88 (m, 2H, H-15), 2.82+2.81 (two overlapping t, J=7.5 Hz, 4H,H-17+H-21), 2.35+2.33 (two overlapping t, J=7.5 Hz, 2H, H-13), 2.07+2.06(two overlapping quint, J=7.5 Hz, 4H, H-18+H-22), 1.84 (quint, J=7.5 Hz,2H, H-14);

¹³C-NMR (300 MHz, D₂O) ppm: δ=175.21 (1C, C-12), 173.82+173.26+173.15(3C, C-9+C-16+C-20), 141.72 (1C, C-3 or C-4), 140.79 (1C, C-3 or C-4),137.02 (1C, C-1), 128.84 (1C, C-6), 124.66 (1C, C-2 or C-5), 124.10 (1C,C-2 or C-5), 54.40 (1C, C-8 or C-10), 54.18 (1C, C-8 or C-10),39.23+39.14 (3C, C-15+C-19+C-23), 36.43 (1C, C-7), 32.63 (1C, C-13),30.91 (2C, C-17+C-21), 23.36 (1C, C-14), 22.51 (2C, C-18+C-22);

HRMS analysis (DCI, CH₄) m/z for C₂₂H₃₄N₄O₇ (calculated=466.243):[M]=466.242.

Preparation of Butyl Esters of mono-GABA-L-DOPA (BL-1023*, Compound 16),bis-GABA-L-DOPA (AN-490, Compound 21) and tri-GABA-L-DOPA (Compound 18)

Preparation of L-DOPA Butyl Ester:

A recent study reported by Reichman et al. showed that L-DOPA butylester has better skin permeability then other esters such as the ethylor octyl esters. Hence the butyl ester of exemplary GABA-L-DOPAconjugates according to some embodiments of the invention, correspondingto the methyl esters Compound 8, Compound 11 and Compound 13 have beenprepared.

Butyl-2-amino-3-(3,4-dihydroxyphenyl)propanoate hydrochloride (Compound14) was obtained in quantitative yield as illustrated in Scheme 6 below.

SOCl₂ (16.5 ml, 228.2 mmol) was added drop wise and under argon to anice-cold and stirred suspension of L-DOPA (5 grams, 25.4 mmol) in n-BuOH(200 ml). The solution was stirred at room temperature for 20 hours andthe solvent was evaporated under reduced pressure. The resulting oilyresidue was dissolved in water and the aqueous phase was washed threetimes with hexane, three times with ether and thereafter lyophilizedunder reduced pressure to afford Compound 12 as an amber solid inquantitative yield (7.3 grams).

¹H-NMR (300 MHz, D₂O) ppm: δ=6.87 (d, J=8.58 Hz, 1H, H-5), 6.75 (d,J=2.52 Hz, 1H, H-2), 6.66 (dd, J=8.58, 2.52 Hz, 1H, H-6), 4.32 (t,J=7.78 Hz, 1H, H-8), 4.18 (t, J=6.67 Hz, 2H, H-10), 3.11 (d, J=7.78 Hz,2H, H-7), 1.54 (m, 2H, H-11), 1.22 (sex, J=8.31 Hz, 2H, H-12), 0.85 (t,J=8.31 Hz, 3H, H-13);

¹³C-NMR (300 MHz, D₂O) ppm: δ=170.33 (1C, C-9), 144.84+144.21 (2C,C-3+C-4), 126.61 (1C, C-1), 122.29 (1C, C-6), 117.46+117.08 (2C,C-5+C-6), 67.68 (1C, C-10), 54.71 (1C, C-8), 35.71 (1C, C-7), 30.22 (1C,C-11), 18.96 (1C, C-12), 13.47 (1C, C-13);

MS analysis (TOF, ES⁺) m/z for C₁₃H₁₉NO₄ (calculated=253.13): [MH⁺]=254;[MH⁺—NH₃]=237.

Preparation of mono- bis- and tri-GABA-L-DOPA Butyl Ester Derivatives(Compounds 16 (BL-1023*), 21 (AN-490) and 18 Respectively):

The mono-, bis- and tris-GABA-L-DOPA butyl ester derivatives, namelyCompound 16, Compound 21 and Compound 18, were prepared as illustratedin Scheme 7 below and described hereinabove for the methyl esters.

Butyl-2-(tert-butyl-3-carbamoylpropylcarbamate)-3-(3,4-dihydroxyphenyl)propanoate(Compound 15) was obtained as foam at 89% yield from Compound 14 and wasused without further purification.

¹H-NMR (300 MHz, CDCl₃) ppm: δ=6.74 (d, J=7.87 Hz, 1H, H-5), 6.69 (bs,1H, H-2), 6.47 (dd, J=7.87, 1.79 Hz, 1H, H-6), 5.00 (bs, 1H, phenol),4.77 (dd, J=13, 6.6 Hz, 1H, H-8), 4.10 (t, J=6.5 Hz, 2H, H-10), 3.05 (m,3H, H-7+H-18), 2.90 (dd, J=13, 6.6 Hz, 1H, H-7), 2.18 (m, 2H, H-16),1.82-1.70 (m, 2H, H-17), 1.59 (m, 2H, H-11), 1.43 (s, 9H, H-22), 1.35(m, 2H, H-12), 0.91 (t, J=7.5 Hz, 3H, H-13);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=173.01 (1C, C-15), 172.00 (1C, C-9),156.97 (1C, C-20), 144.27 (1C, C-3 or C-4), 143.78 (1C, C-3 or C-4),127.85 (1C, C-1), 121.08 (1C, C-6), 116.42 (1C, C-2 or C-5), 115.33 (1C,C-2 or C-5), 80.07 (1C, C-21), 65.64 (1C, C-10), 53.76 (1C, C-8), 39.92(1C, C-18), 37.21 (1C, C-7), 33.46 (1C, C-16), 30.59 (1C, C-11), 28.51(3C, C-22), 26.07 (1C, C-17), 19.17 (1C, C-12), 13.76 (1C, C-13);

MS analysis (TOF, ES⁺) m/z for C₂₂H₃₄N₂O₇ (calculated=438.24: [MH⁺]=439;[M+Na⁺]=461; [MH⁺-Bu]=383; [MH⁺-Bu-CO₂]=340.

Butyl-2-(4-Aminobutanamide)-3-(3,4-dihydroxyphenyl)propanoatehydrochloride (Compound 16) was obtained from Compound 15 to afford asolid which was washed with ether and dried under reduced pressure. Thesolid was dissolved in water and washed three times with ether. Theaqueous phase was evaporated and lyophilized to afford the Compound 16as yellowish solid at 85% yield.

Melting point (mp): 77° C.;

¹H-NMR (300 MHz, D₂O) ppm: δ=6.84 (d, J=7.77 Hz, 1H, H-5), 6.74 (d,J=2.43 Hz, 1H, H-2), 6.64 (dd, J=7.77, 2.43 Hz, 1H, H-6), 4.55 (dd,J=8.78, 7.02 Hz, 1H, H-8), 4.08 (t, J=7.02 Hz, 2H, H-10), 3.00 (m, 1H,H-7), 2.92 (m, 3H, H-7+H-18), 2.34 (t, J=6.99 Hz, 2H, H-16), 1.85(quint, J=6.99 Hz, 2H, H-17), 1.52 (m, 2H, H-11), 1.21 (sex, J=6.99 Hz,2H, H-12), 0.84 (t, J=6.99 Hz, 3H, H-13);

¹³C-NMR (300 MHz, D₂O) ppm: δ=175.12 (1C, C-15), 174.18 (1C, C-9),144.46+143.47 (2C, C-3+C-4), 129.43 (1C, C-1), 122.01 (1C, C-6),117.33+116.74 (2C, C-2+C-5), 66.76 (1C, C-10), 55.11 (1C, C-8), 39.18(1C, C-16 or C-18), 36.58 (1C, C-16 or C-18), 32.52 (1C, C-7), 30.31(1C, C-11), 23.39 (1C, C-17), 19.00 (1C, C-12), 13.44 (1C, C-13);

HRMS analysis (DCI, CH₄) m/z for C₁₇H₂₆N₂O₅ (calculated=338.184):[MH⁺]=339.166.

2-[(N-4-Aminobutyramido)-3-(3,4-bis-(4-tert-butoxycarbonyl-aminobutyryloxy)phenyl]propionatebutyl ester (Compound 17) was obtained as a white solid from Compound 16after recrystallization from DCM:ether at 77% yield.

Melting point (mp): 125-126° C.;

¹N-NMR (300 MHz, CDCl₃) ppm: δ=7.08 (d, J=8.22 Hz, 1H, H-5), 7.01-6.98(two broad peaks, 2H, H-2+H-6), 4.82 (m, 1H, H-8), 4.1 (td, J=6.02, 3Hz, 2H, H-10), 3.2 (t, J=7.52 Hz, 4H, H-26+H-34), 3.13-3.00 (m, 4H,H-7+H-18), 2.58 (t, J=7.90 Hz, 4H, H-24+H-32), 2.20 (t, J=6.60 Hz, 2H,H-16), 1.90 (m, 4H, H-25+H-33), 1.74 (m, 2H, H-17), 1.60-1.53 (m, 2H,H-11), 1.44+1.41 (two s, 27H, H-22+H-30+H-38), 1.39-1.29 (m, 2H, H-12),0.90 (t, J=6.60 Hz, 3H, H-13);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=172.61 (1C, C-15), 171.54+170.59 (3C,C-9+C-23+C-31), 156.42+156.15+156.12 (3C, C-20+C-28+C-36), 141.69 (1C,C-3 or C-4), 140.83 (1C, C-3 or C-4), 135.03 (1C, C-1), 127.14 (1C,C-6), 124.40 (1C, C-2 or C-5), 123.30 (1C, C-2 or C-5), 79.13 (3C,C-21+C-29+C-37), 65.42 (1C, C-10), 53.02 (1C, C-8), 39.71 (3C,C-18+C-26+C-34), 37.03 (1C, C-7), 33.23 (1C, C-16), 31.20+30.41 (3C,C-11+C-24+C-32), 28.39 (9C, C-22+C-30+C-38), 26.03 (1C, C-17), 25.26(2C, C-25+C-33), 18.99 (1C, C-12), 13.61 (1C, C-13);

MS analysis (TOF, ES⁺) m/z for C₄₀H₆₄N₄O₁₃ (calculated=808.45):[MH⁺]809; [MH⁺-Boc]=709.

2-[(N-4-Aminobutyramido)-3-(3,4-bis-(4-amino-butyroyloxy)phenyl]propanoatebutyl ester tri-hydrochloride (Compound 18) was obtained from Compound17 as a yellowish solid in quantitative yields.

Melting point (mp): 74-75° C.;

¹H-NMR (300 MHz, D₂O) ppm: δ=7.27-7.16 (m, 3H, H-2+H-5+H-6), 4.68 (dd,J=9.0, 6.5 Hz, 1H, H-8), 4.10 (t, J=5.7 Hz, 2H, H-10), 3.24-2.87 (m, 8H,H-7+H18+H-23+H-28), 2.81 (td, J=7.4, 1.8 Hz, 4H, H-21+H-26), 2.34 (td,J=7.6, 2.0 Hz, 2H, H-16), 2.06 (m, 4H, H-22+H-27), 1.85 (quint, J=7.6Hz, 2H, H-17), 1.53 (m, 2H, H-11), 1.27 (m, 2H, H-12), 0.86 (t, J=7.5Hz, 3H, H-13);

¹³C-NMR (300 MHz, D₂O) ppm: δ=175.18 (1C, C-15), 173.60 (1C, C-9),173.17+173.06 (2C, C-20+C-25), 141.73 (1C, C-3 or C-4), 140.81 (1C, C-3or C-4), 136.86 (1C, C-1), 128.78 (1C, C-6), 124.62 (1C, C-2 or C-5),124.13 (1C, C-2 or C-5), 66.92 (1C, C-10), 54.50 (1C, C-8), 39.25+39.13(3C, C-18+C-23+C-28), 36.47 (1C, C-7), 32.60 (1C, C-16), 30.91 (2C,C-21+C-26), 30.32 (1C, C-11), 23.38 (1C, C-17), 22.15 (2C, C-22+C-27),19.05 (1C, C-12), 13.50 (1C, C-13);

HRMS analysis (DCI, CH₄) m/z for C₂₅H₄₀N₄O₇ (calculated=508.290):[M]=508.294; [C₁₇H₂₇N₂O₅ ⁺]=339.187.

tert-Butyl-1-(butoxycarbonyl)-2-(3,4-dihydroxyphenyl)ethylcarbamate(Compound 19) was obtained at 85% yield from Compound 14 and the crudewas used in the next step without further purification.

¹H-NMR (300 MHz, CDCl₃) ppm: δ=6.73 (d, J=8.1 Hz, 1H, H-5), 6.65 (bs,1H, H-2), 6.49 (bd, J=8.1 Hz, 1H, H-6), 5.12 (d, J=8.4 Hz, 1H), 4.48 (m,1H, H-8), 4.09 (t, J=6.30 Hz, 2H, H-10), 2.95 (m, 2H, H-7), 1.55 (m, 2H,H-11), 1.40 (s, 9H, H-17), 1.32 (m, 2H, H-12), 0.89 (t, J=7.2 Hz, H-13);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=172.58 (1C, C-9), 155.70 (1C, C-15),144.17 (1C, C-3 or C-4), 143.29 (1C, C-3 or C-4), 128.17 (1C, C-1),121.41 (1C, C-6), 116.24 (1C, C-2 or C-5), 115.35 (1C, C-2 or C-5),80.51 (1C, C-16), 65.55 (1C, C-10), 54.84 (1C, C-8), 37.89 (1C, C-7),30.55 (1C, C-11), 28.36 (3C, C-17), 19.11 (1C, C-12), 13.72 (1C, C-13).

tert-Butyl-1-(butoxycarbonyl)-2-(3,4-[di-tert-butyl-4-butoxyphenylcarbamate])-dihydroxyphenyl)ethylcarbamate (Compound 20) was obtainedfrom Compound 19 after recrystallization from DCM:ether which afforded awhite solid, which was further filtered and recrystallized fromether:hexane to afford a second batch which wad combined to a totalyield of 75%.

Melting point (mp): 85-86° C.;

¹H-NMR (300 MHz, CDCl₃) ppm: δ=7.10 (d, J=8.1 Hz, 1H, H-5), 7.01 (d,J=1.8 Hz, 1H, H-2), 6.98 (m, 1H, H-6), 5.05 (d, J=7.4 Hz, 1H), 4.91 (bs,1H), 4.52 (m, 1H, H-8), 4.07 (t, J=6.6 Hz, 2H, H-10), 3.20 (t, J=6.8 Hz,4H, H-21+H-29), 3.05 (m, 2H, H-7), 2.55 (t, J=7.4 Hz, 4H, H-19+H-27),1.88 (quint, J=7.2 Hz, 4H, H-20+H-28), 1.54 (m, 2H, H-11), 1.42 (s, 18H,H-25+H-33), 1.40 (s, 9H, H-17), 1.30 (m, 2H, H-12), 0.88 (t, J=7.5 Hz,3H, H-13);

¹³C-NMR (300 MHz, CDCl₃) ppm: δ=171.73 (1C, C-9), 170.64+170.49 (2C,C-18+C-26), 156.17 (3C, C-15+C-23+C-31), 141.84 (1C, C-3 or C-4), 140.97(1C, C-3 or C-4), 135.10 (1C, C-1), 127.39 (1C, C-6), 124.34 (1C, C-2 orC-5), 123.40 (1C, C-2 or C-5), 80.05+79.45 (2C, C-16+C-24+C-32), 65.47(1C, C-10), 54.34 (1C, C-8), 39.93 (2C, C-21+C-29), 37.76 (1C, C-7),31.33 (2C, C-19+C-27), 30.51 (1C, C-11), 28.49 (6C, C-25+C-33), 28.49(3C, C-17), 25.42+25.39 (2C, C-20+C-28), 19.09 (1C, C-12), 13.70 (1C,C-13);

MS analysis (TOF, ES⁺) m/z: for C₃₆H₅₇N₃O₁₂ (calculated=723.39):[MH⁺]=724; [M+Na⁺]=746; [MH⁺-Boc]=624.

Butyl-2-amino-3-(3,4-phenyl-di[4-aminobutanoate])propanoatetri-hydrochloride (Compound 21) was obtained from Compound 20 afterrecrystallization from MeOH:ether to afford a white solid at 88% yield.

Melting point (mp): 175-176° C.;

¹H-NMR (300 MHz, D₂O) ppm: δ=7.36 (d, J=8.22 Hz, 1H, H-5), 7.33 (d,J=1.80 Hz, 1H, H-2), 7.29 (dd, J=8.22, 1.80 Hz, 1H, H-6), 4.46 (t,J=6.67 Hz, 1H, H-8), 4.24 (t, J=6.67 Hz, 2H, H-10), 3.34 (d, J=6.67 Hz,2H, H-7), 3.15 (bt, 4H, H-17+H-21), 2.863+2.857 (t, J=7.19 Hz, 4H,H-15+H-19), 2.10 (quint, J=7.51 Hz, 4H, H-16+H-20), 1.60 (m, 2H, H-11),1.29 (sex, J=7.78 Hz, 2H, H-12), 0.89 (t, J=7.78 Hz, 3H, H-13);

¹³C-NMR (300 MHz, D₂O) ppm: δ=173.20 (2C, C-14+C-18), 169.98 (1C, C-9),142.11+141.55 (2C, C-3+C-4), 134.37 (1C, C-1), 129.19 (1C, C-6),125.11+124.82 (2C, C-2+C-5), 67.87 (1C, C-10), 54.42 (1C, C-8), 39.14(2C, C-17+C-21), 35.57 (1C, C-7), 30.93 (2C, C-15+C-18), 30.21 (1C,C-11), 22.50 (2C, C-16+C-20), 19.00 (1C, C-12), 13.51 (1C, C-13);

HRMS analysis (DCI, CH₄) m/z: for C₂₁H₃₃N₃O₆ (calculated=423.237):[C₁₇H₂₇N₂O₅ ⁺]=339.190 (no molecular peak, only fragments weredetected).

Synthesis of C3 or C4 Mono GABA-L-DOPA Butyl Ester Derivative (Compounds21a, and 21b)

The bis-GABA ester of L-DOPA butyl ester (compound 21, AN-490), containsthree ammonium hydrochloride moieties, two of them belong to GABAmoieties in the conjugate, and the other to L-DOPA. The compound, havingthree basic amino functionalities in the buffered in vivo environment,may be found in equilibrium with some protonated species which may betoo polar in order to cross the BBB. The relatively high molecularweight of AN-490 (533 grams/mol) may also diminish the effectiveness ofdrug diffusion.

Therefore, mono-GABA-L-DOPA butyl ester derivatives, in which a singleGABA moiety is linked to only one hydroxyl group of L-DOPA, either atthe C-4 or at the C-3 position, yet leaving the n-butyl ester on thecarboxylic moiety of L-DOPA unchanged, were synthesized. These compoundshave a lower molecular weight (411 grams/mol), as compared to AN-490 andhave only two basic amino groups, and are thus potentially moreapplicable for clinical use.

The synthetic strategy for a mono-GABA-L-DOPA butyl ester was similar tothat used for preparing AN-490 (Compound 21) described hereinabove, withthe required stoichiometric adjustments. Thus, N-protected L-DOPAn-butyl ester was reacted with one equivalent of carbodiimide-activatedN-protected-GABA, as illustrated in Scheme 8 below.

L-DOPA was converted to the corresponding butyl ester 14 as describedhereinabove, using thionyl chloride in n-butyl alcohol, and the obtainedbutyl ester was then N-protected by treatment with Boc.

The Boc protecting group was selected upon undesired results that wereobtained while using N-carboxybenzyl (CBZ) protected Compound 14.

Thus, CBZ-protected Compound 14 was catalytically hydrogenated, via anaddition-elimination reaction, so as to cleave the CBZ group and yieldfree L-DOPA and 2-pyrrolidone, the cyclic lactam of GABA.

Boc-protection of Compound 14 was performed by adding to a solution ofL-DOPA alkyl ester (1 equivalent) in dioxane and water (2:1 v/v) 1 MNaOH solution (1 equivalent). After 10 minutes, di-tert-butyldicarbonate (1 equivalent) was added and the reaction mixture wasstirred at room temperature over night. The solvent was thereafterevaporated and the residue was diluted with water and EtOAc. The layerswere separated and the organic layer was washed with 1 M KHSO₄ (×3), 1 MNaHCO₃ (×3) and brine (×3), dried over Na₂SO₄ and evaporated to give thecrude Compound 19.

¹H NMR (300 MHz, CDCl₃) ppm: δ=0.84 (t, J=7.35 Hz, 3H, H-13), 1.27 (m,2H, H-12), 1.37 (s, 9H, H-17), 1.52 (m, 2H, H-11), 2.76-2.96 (m, [AB ofABX system], 2H, H-7), 4.04 (m, 2H, H-10), 4.27-4.48 (m [X of ABXsystem], 1H, H-8), 5.23 (d, J=8.40 Hz, 1H, H-14), 6.46 (dd,J_(ortho)=8.10 Hz, J_(meta)=1.80 Hz, 1H, H-6), 6.64 (d, J_(meta)=1.80Hz, 1H, H-2), 6.71 (d, J_(ortho)=8.10 Hz, 1H, H-5), 6.94 and 7.05 (twobr s, 2H, 3-OH+4-OH);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=13.53 (C-13), 18.92 (C-12), 28.18 (C-17),30.34 (C-11), 37.52 (C-7), 54.76 (C-8), 65.40 (C-10), 80.39 (C-16),115.30 and 116.19 (C-2+C-5), 121.14 (C-6), 127.86 (C-1), 143.24 and144.17 (C-3+C-4), 155.76 (C-15), 172.56 (C-9);

MS (CI+): m/z=354.194 ([MH]^(•+), 4.66), 298.129 ([MH—C₄H₈]^(•+),31.85), 254.128 ([MH—C₅H₈O₂]^(•+), 83.98), 236.090 ([C₁₃H₁₆O₄]^(•+),100.00);

HRMS calcd. for C₁₈H₂₈NO₆ ([MH]^(•+), DCI, CH₄) 354.1917. found354.1943.

The crude product was used in the next step without furtherpurification.

The resulting N-Boc-protected butyl ester Compound 19 was mixed withEDC-activated N-Boc-GABA as follows:

To an ice-cold stirred solution of N-protected-GABA (1 equivalent) inDMF were added EDC (1.1 equivalent) and HOBt (1.1 equivalent). After 1hour, the ice bath was removed and N-protected-L-DOPA butyl ester(Compound 19, 1 equivalent) and triethanolamine (TEA, 3 equivalents)were added. The mixture was stirred over night at room temperature, thesolvent was thereafter evaporated, and the residue was dissolved inEtOAc and water. The layers were separated, and the organic phase waswashed with 1 M KHSO₄ (×3), 1 M NaHCO₃ (×3), and brine (×3), dried overNa₂SO₄ and evaporated, to afford the “bis-ester” Compound 20c and anisomeric mixture of the mono-substituted 3-O- and 4-O-acylated catechols(Compounds 20a and 20b).

The crude product was chromatographed twice (using as eluents CHCl₃:MeOH80:1 v/v and then hexane:EtOAc 3:1 v/v), to provide a mixture ofCompounds 20a and 20b as colorless oil in 37% yield. The ratio of theisomers in the mixture was determined based on ¹H-NMR integrations ofthe H-2 in both isomers. The NMR data assignment was aided by severaltwo-dimensional spectra including COSY, HMQC and HMBC analyses.

Unless otherwise indicated, the chemical shifts are assigned to bothisomers. Superscript notations ^(a)/^(b) refer to nuclei assigned to thecorresponding isomers in the mixture.

¹H NMR (600 MHz, CDCl₃) ppm: δ=0.91 (t, J=7.20 Hz, 2.4H, H-13^(b)), 0.92(t, J=7.50 Hz, 3H, H-13^(a)), 1.30-1.37 (m, ˜4H, H-12), 1.42 and 1.43(two overlapping singlets, ˜16H, H-17), 1.46 (s, ˜16H, H-25), 1.53-1.63(m, ˜4H, H-11), 1.85 (br quint, 3.6H, H-20), 2.62-2.64 (m, 3.6H, H-19),2.82-3.06 (m, 3.6H, H-7), 3.28 (br q, 3.6H, H-21), 4.09 (t, J=6.60 Hz,1.6H, H-10^(b)), 4.11 (t, J=6.30 Hz, 2H, H-10^(a)), 4.50 (br q, ˜0.8H,H-8^(b)), 4.53 (br q, 1H, H-8^(a)), 4.79 (br t, 1.8H, H-22), 5.00 (br d,˜1.8H, H-14), 6.63 (dd, J_(ortho)=8.40 Hz, J_(meta)=1.80 Hz, 1H,H-6^(a)), 6.76 (br s, 0.8H, H-2^(b)), 6.82 (br s, 1H, H-2^(a)), 6.88 (m,0.8H, H-6^(b)), 6.89 (d, J_(ortho)=8.40 Hz, 1H, H-5^(a)), 6.95 (d,J_(ortho)=7.80 Hz, 0.8H, H-5^(b)).

¹³C NMR (determined by HMBC analysis; 150 MHz, CDCl₃) ppm: δ=13.66(C-13), 13.05 (C-12^(b)), 19.07 (C-12^(a)), 25.99 (C-20), 28.32 (C-17),28.40 (C-25), 29.99 (C-19), 30.52 (C-11), 37.44 (C-7^(b)), 37.85(C-7^(a)), 38.20 (C-21), 54.38 (C-8^(a)), 54.53 (C-8^(b)), 65.25(C-10^(b)), 65.29 (C-10^(a)), 79.90, 80.30 and 81.97 (C-16+C-24), 117.87(C-5^(b)), 118.79 (C-2^(a)), 120.75 (C-6^(a)), 122.64 (C-5^(a)), 123.48(C-2^(b)), 127.93 (C-6^(b)), 135.16 (C-1^(a)), 136.9 (C-4^(a)), 137.81(C-3^(b)), 147.5 (C-4^(b)), 148.5 (C-3^(a)), 155.19 (C-15), 156.94(C-23), 171.27 (C-18), 171.96 (C-9).

MS (CI+): m/z=539.296 ([MH]^(•+), 4.23), 483.131 ([MH—C₄H₈]^(•+), 4.46),427.080 ([MH—C₄H₈—C₄H₈]^(•+), 10.81), 383.107 (MH—C₄H₈—C₅H₈O₂]^(•+),71.89).

HRMS calcd. for C₂₇H₄₃N₂O₉ ([MH]^(•+), DCI, CH₄) 539.2969. found539.2961.

Removal of the N-Boc protecting groups of compounds 20a and 20b wasperformed as follows (see, Scheme 9 below): To an ice-cold solution ofthe obtained mixture of Compounds 20a and 20b as well as the undesiredCompound 20c in EtOAc, a freshly prepared solution of 4 N HCl in EtOAcwas added (obtained by addition of a known amount of acetyl chloride toan ice cold solution of an equivalent amount of EtOH in EtOAc). The icebath was removed after 1 hour, and the solution was allowed to warm toroom temperature. Once the reaction was completed (as monitored by TLCfor complete consumption of Compounds 20a and 20b, the solvent wasevaporated to give Compounds 21a and 21b.

Remnants of the starting Boc-protected L-DOPA (Compound 19) were removedby flash chromatography. Subsequently, the monoesters' mixture ofCompounds 21a and 21b was isolated from the bis-ester Compound 21c in37% yield by a consecutive column chromatography in a different eluent.

¹H NMR analysis of an aliquot in D₂O showed about 91% of the isomersmixture of Compounds 21a and 21b in an approximately 1.16:1 ratio,respectively (as presented hereinafter), along with about 4.5% L-DOPAbutyl ester hydrochloride (Compound 14) and about 4.5% GABA.

The ratio of isomers was determined based on ¹H-NMR integrations of theH-2 in both isomers. Unless otherwise indicated, the chemical shifts areassigned to both isomers. Superscript notations ^(a)/^(b) refer tonuclei assigned to the corresponding isomers in the mixture.

¹H NMR (300 MHz, D₂O) ppm: δ=0.87 (t, J=7.35 Hz, 5.6H, H-13), 1.23-1.33(m, ˜4H, H-12), 1.53-1.63 (m, 3.7H, H-11), 2.10 (quint, J=7.43 Hz, 3.7H,H-16), 2.85 (t, J=7.20 Hz, 3.7H, H-15), 3.14 (m, 3.7H, H-17), 3.24 (m,3.7H, H-7), 4_(:)22 (t, J=6.60 Hz, 1.7H, H-10^(b)), 4.23 (t, J=6.60 Hz,2H, H-10^(a)), 4.37 (t, J=6.90 Hz, 0.9H, H-8^(b)), 4.41 (t, J=6.90 Hz,1H, H-8^(a)), 6.88 (dd, J_(ortho)=8.10 Hz, J_(meta)=1.80 Hz, 1H,H-6^(a)), 6.94 (d, J_(meta)=1.80 Hz, 1H, H-2^(a)), 7.03 (d,J_(meta)=1.80 Hz, 0.9H, H-2^(b)), 7.05 (d, J_(ortho)=8.10 Hz, 1H,H-5^(a)), 7.11 (dd, J_(ortho)=8.10 Hz, J_(meta)=1.80 Hz, 0.9H, H-6^(b)),7.13 (d, J_(ortho)=8.10 Hz, 0.9H, H-5^(b)).

¹³C NMR (75 MHz, D₂O) ppm: δ=13.50 (C-13), 19.05 (C-12), 22.63 (C-16),30.31 (C-15), 31.02 (C-11), 35.45 (C-7^(b)), 35.97 (C-7^(a)), 54.64(C-8^(a)), 54.72 (C-8^(b)), 67.90 (C-10), 118.41, 118.69, 122.45, 124.29and 124.51 (Ar—CH), 127.08 (C-1^(a or b)), 129.37 (Ar—CH), 134.30(C-1^(a or b)), 138.27 and 138.75 (C-4^(a)+C-3^(b)), 147.63 and 148.26(C-3^(a)+C-4^(b)), 170.33 (C-9), 174.37 (C-14).

MS (CI+): m/z=337.172 ([M-H]^(•+), 8.17), 254.142([ArCH₂CH(NH₃)CO₂Bu]^(•+), 100.00), 86.053 ([NH₂(CH₂)₃CO]^(•+), 42.65).

HRMS calcd. for C₁₇H₂₅N₂O₅ ([M—H]^(•+), DCI, CH₄) 337.1763. found539.1721.

Preparation of Benzenesulfonic Acid Addition Salts

The hydrochloride salts of Compounds 6, 8a, 11, 13, 14, 16, 18 and 21were identified as hygroscopic. Hence, a corresponding benzenesulfonateaddition salt (Compound 22) was prepared as illustrated in Scheme 10 asa model to evaluate its hygroscopicity. The basis for this assumptionwas that the addition salt of an aromatic acid would be less hygroscopicthan that of the corresponding hydrochloric acid addition salt. Thus,the butyl ester, Compound 20 was Boc-deprotected with 3 equivalents ofbenzenesulfonic acid in 1,2-dichloromethane.

The isolated salt, Compound 22, was found to be much less hygroscopicthan the hydrochloride salt.

Butyl-2-amino-3-(3,4-phenyl-di[4-aminobutanoate])propanoatetri-benzenesulfonate salt (Compound 22) was prepared by addingbenzenesulfonic acid (496 mg, 3.14 mmol) to a stirred solution ofCompound 20 (751 mg, 1.04 mmol) in dry DCM (30 ml). After 4 hours waterwas added and the aqueous phase was separated, washed with DCM andevaporated to dryness. Recrystallization from MeOH:ether affordedCompound 22 as a brownish solid (707 mg, 76% yield).

Melting point (mp): 128° C.;

¹H-NMR (300 MHz, D₂O) ppm: δ=7.81-7.77 (m, 6H, H-22+H-26), 7.57+7.47 (m,9H, H-23+H-24+H-25), 7.26-7.14 (m, 3H, H-2+H-5+H-6), 4.35 (t, J=7.1 Hz,1H, H-8), 4.14 (t, J=6.3 Hz, 2H, H-10), 3.24 (d, J=7.1 Hz, 1H, H-7),3.02 (m, 4H, H-17+H-21), 2.73 (t, J=7.2 Hz, 3H, major rotamer,H-15+H-19), 2.47 (t, J=7.2 Hz, 1H, minor rotamer, H-15+H-19), 2.05-1.89(m, 4H, H-16+H-21), 1.50 (m, 2H, H-11), 1.20 (m, 2H, H-12), 0.82 (t,J=7.5 Hz, 3H, H-13);

¹³C-NMR (300 MHz, D₂O) ppm: δ=173.05 (2C, C-14+C-18), 169.93 (1C, C-9),142.86 (3C, C-27), 141.99+141.43 (2C, C-3+C-4), 134.28 (1C, C-1), 132.10(3C, C-24), 129.52 (6C, C-23+C-25), 129.06 (1C, C-6), 125.87 (6C,C-22+C-26), 124.96+124.70 (2C, C-3+C-4), 67.77 (1C, C-10), 54.32 (1C,C-8), 39.04 (2C, C-17+C-21), 35.56 (1C, C-7), 30.78 (2C, C-15+C-18),30.13 (1C, C-11), 22.42 (2C, C-16+C-20), 18.93 (1C, C-12), 13.42 (1C,C-13);

HRMS analysis (DCI, CH₄): m/z for C₂₁H₃₃N₃O₆ (calculated=423.503):[MH⁺]=422.231.

Preparation of L-DOPA-OCH₂O-GABA Derivative (Compound 33)

An alternative approach to couple GABA with L-DOPA, is by takingadvantage of the carboxylic acid functionality of L-DOPA for the purposeof acylation, rather than its hydroxyls. While in the L-DOPA mono-GABAester Compounds 21a and 21b the carboxylic acid moiety of L-DOPA wasesterified with n-butanol to reduce the hydrophilicity, theacyloxymethyl derivative 33 (see, Scheme 11 below) encompasses thecarboxylic moieties of L-DOPA and GABA attached via a labile —OCH₂O—linker. Linking L-DOPA and GABA via an oxyalkylester linkage, such asoxymethylester linkage, further advantageously provides conjugate thatcan release formaldehyde as an additional metabolite upon in vivohydrolytic cleavage of the acyloxymethyl ester derivative, alongside ofL-DOPA and GABA (see, Scheme 11). Formaldehyde was shown to exhibitbeneficial therapeutic effect.

The following synthetic pathway for preparing Compound 33 was devised(see, Scheme 12 below), while taking into consideration the effect andremoval selectivity of the selected protecting groups.

L-DOPA was converted into its corresponding alkyl ester, followed byBoc-protection of the amino moiety. Dibenzylation of the L-DOPAhydroxyls was thereafter efficiently accomplished, and was followed byhydrolysis of the alkyl ester so as to uncovers the free acidfunctionality. Base-mediated acyloxymethylation with N-Boc-GABA-OCH₂Clwas then performed, followed by deprotection of the L-DOPA and aminofunctions by hydrogenolysis and acidification, respectively, so as toafford the desired product.

L-DOPA methyl ester hydrochloride (Compound 6) was prepared from L-DOPAusing thionyl chloride in methanol. N-Boc-protection of Compound 6,employing di-tert-butyl dicarbonate in the presence of aqueous NaOHsolution led to a mixture of Compound 26 and two N,O-Boc isomersCompounds 27a and 27b (see, Scheme 13), resulting from a furtheracylation of one of the L-DOPA hydroxyl groups. The N-Boc-protection ofCompound 6 was performed as follows: To a solution of L-DOPA methylester (Compound 6, 1 equivalent) in dioxane and water (2:1 v/v) wasadded 1 M NaOH solution (1 equivalent). After 10 minutes, di-tert-butyldicarbonate (1 equivalent) was added and the reaction was stirred atroom temperature over night. The solvent was thereafter evaporated andthe residue was diluted with water and EtOAc. The layers were separatedand the organic layer was washed with 1 M KHSO₄ (×3), 1 M NaHCO₃ (×3)and brine (×3), dried over Na₂SO₄ and evaporated to give the crudeproduct Compound 26 and the two N,O-Boc isomers Compounds 27a and 27b.The crude product was chromatographed (using hexane:EtOAc 2:1 v/v aseluent, R_(f)=0.12), to remove the isomeric N,O-Boc-protectedby-products, giving Compound 26 as a white solid (30% yield).

An alternative synthesis method for obtaining Compound 26 was alsosuccessfully performed. To a solution of Compound 6 (4.00 grams, 16.15mmol) in water (35 ml) were added NaHCO₃ (2.71 grams, 32.30 mmol). Aftera few minutes, a solution of di-tert-butyl dicarbonate (3.52 grams,16.15 mmol) in THF (36 ml) was added. The mixture was stirred at roomtemperature for 19 hours, and thereafter concentrate under vacuum toremove THF. Water and EtOAc were added and the layers were separated.The organic phase was washed with 1 N HCl (×2) and water (×2), driedover Na₂SO₄ and evaporated to give the crude product (4.58 grams), ofwhich 1.86 grams were purified by chromatography (using hexane:EtOAc 2:1v/v as eluent) to give Compound 26 as a white solid (1.29 gram, 69%chromatographic yield).

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.36 (s, 9H, H-14), 2.78-2.96 (m [AB ofABX system], 2H, H-7), 3.64 (s, 3H, H-10), 4.46 (m [X of ABX system],1H, H-8), 5.22 (br d, J=8.40 Hz, 1H, H-11), 6.65 (br dd, J_(ortho)=8.10Hz, 1H, H-6), 6.75 (br d, 1H, H-2), 6.87 (d, J_(ortho)=8.10 Hz, 1H,H-5), 6.98 (br s, 2H, 3-OH+4-OH);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=28.25 (C-14), 37.58 (C-7), 52.35 (C-10),54.72 (C-8), 80.51 (C-13), 115.38 and 116.17 (C-2+C-5), 122.24 (C-6),127.94 (C-1), 143.32 and 144.22 (C-3+C-4), 155.76 (C-12), 172.90 (C-9);

MS (CI+): m/z=312.143 ([MH]^(•+), 55.43), 256.069 ([MH—C₄H₈]^(•+),73.31), 212.093 ([MH—C₅H₈O₂]^(•+), 100.00);

HRMS calcd. for C₁₅H₂₂NO₆ ([MH]^(•+), DCI, CH₄) 312.1447. found312.1431.

Reagents and conditions: (a) SOCl₂, MeOH, 0° C. to rt; (b) Boc₂O, 1 MNaOH, H₂O/dioxane, rt; (c) Boc₂O, NaHCO₃, H₂O/THF, rt.

Replacement of NaOH by NaHCO₃ (see, b or c in Scheme 13) resulted in afewer amount of side-products, so that the crude product consistedalmost exclusively of the N-protected Compound 26. The variation inproduct distribution reflects the competition between N- andO-acylation, as derived from the relative proximity of the acidityconstants predicted for the ammonium and first phenolic ionizations (itis noted that L-DOPA exhibits two overlapping acidity constants at 0.16ionic strength and 25° C.: pK_(OH)=8.97, pK_(NH3)=9.42). However, thebase strength seems to play a significant role in the productdistribution: in a weak basic medium (NaHCO₃) the N-Boc productpredominates, whereas in a strong alkaline medium (NaOH) the formationof a phenolate anion becomes more favorable, hence leading to theN,O-Boc product. In any case, the amino group takes precedence over thephenolate regarding tert-butoxycarbonylation, due to its betternucleophilicity.

Subsequently, the N-Boc-L-DOPA methyl ester, Compound 26, was treatedwith K₂CO₃, NaI, an excess of benzyl bromide and a catalytic amount ofn-Bu₄N⁺Br⁻ under reflux to afford Compound 28 (see, scheme 14 below).

The dibenzylation of Compound 26 was successfully performed as follows:To a solution of N-Boc-L-DOPA methyl ester (Compound 26, 1 equivalent)in dry acetone under N₂ atmosphere were added K₂CO₃ (2.2 equivalents),NaI (0.2 equivalents), n-Bu₄N⁺Br⁻ (0.2 equivalents) and benzyl bromide(3 equivalents). The reaction mixture was refluxed for 4 hours andthereafter evaporated under vacuum. Dichloromethane (DCM) and water wereadded, the layers separated and the aqueous phase was washed to with DCM(×2). The combined organic layer was washed with water (×2), dried overNa₂SO₄ and evaporated to give the crude product Compound 28.

The crude product was purified by column chromatography (usinghexane:EtOAc 4:1 v/v as eluent, R_(f)=0.20) to afford the desiredCompound 28 as a white solid (72% yield).

Melting point (mp): 102-105° C. [lit.⁴ mp 112° C.].

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.44 (s, 9H, H-14), 2.93-3.05 (m [AB ofABX system], 2H, H-7), 3.65 (s, 3H, H-10), 4.54 (m [X of ABX system],1H, H-8), 4.98 (br d, J=8.10 Hz, 1H, H-11), 5.13 (s, 4H, H-15+H-20),6.65 (dd, J_(ortho)=8.10 Hz, J_(meta)=1.80 Hz, 1H, H-6), 6.75 (d,J_(meta)=2.10 Hz, 1H, H-2), 6.87 (d, J_(ortho)=8.10 Hz, 1H, H-5),7.29-7.47 (m, 10H, H-17+H-18+H-19+H-22+H-23+H-24);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=28.42 (C-14), 37.88 (C-7), 52.24 (C-10),54.52 (C-8), 71.44 (C-15+C-20), 80.02 (C-13), 115.23 and 116.31(C-2+C-5), 122.40 (C-6), 127.37, 127.43, 127.86, 127.92, 128.55 and128.58 (C-17+C-18+C-19+C-22+C-23+C-24), 129.35 (C-1), 137.29 and 137.40(C-16+C-21), 148.21 and 149.01 (C-3+C-4), 155.17 (C-12), 172.43 (C-9);

MS (CI+): m/z=491.230 ([M]^(•+), 11.02), 392.176 ([MH—C₅H₈O₂]^(•+),22.28);

HRMS calcd. for C₂₉H₃₃NO₆ ([M]^(•+), DCI, CH₄) 491.2308. found 491.2351.

Dibenzylated N-Boc-L-DOPA butyl ester, Compound 29, was prepared fromCompound 19, similarly to the methyl ester analog, and was subjected tosaponification under the same conditions, but also afforded the methylester Compound 28.

The crude product was chromatographed (using hexane:EtOAc 4:1 v/v aseluent, R_(f)=0.32) to give Compound 29 as a white solid (63% yield).

Melting point (mp): 96-98° C.

¹H NMR (300 MHz, CDCl₃) ppm: δ=0.91 (t, J=7.35 Hz, 3H, H-13), 1.24-1.38(m, 2H, H-12), 1.42 (s, 9H, H-17), 1.46-1.65 (m, 2H, H-11), 2.99 (d,J=5.40 Hz, 2H, H-7), 3.96-4.12 (m, 2H, H-10), 4.44-4.58 (m [X of ABXsystem], 1H, H-8), 4.95 (br d, J=12.00 Hz, 1H, H-14), 5.12 (s, 4H,H-18+H-23), 6.65 (dd, J_(ortho)=8.10 Hz, J_(meta)=1.80 Hz, 1H, H-6),6.75 (d, J_(meta)=1.80 Hz, 1H, H-2), 6.86 (d, J_(ortho)=8.10 Hz, 1H,H-5), 7.26-7.46 (m, 10H, H-20+H-21+H-22+H-25+H-26+H-27);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=13.81 (C-13), 19.19 (C-12), 28.47 (C-17),30.66 (C-11), 38.01 (C-7), 54.60 (C-8), 65.32 (C-10), 71.52 (C-18+C-23),80.00 (C-16), 115.29 and 116.43 (C-2+C-5), 122.51 (C-6), 127.40, 127.50,127.90, 127.95 and 128.61 (C-20+C-21+C-22+C-25+C-26+C-27), 129.53 (C-1),137.34 and 137.49 (C-19+C-24), 148.28 and 149.09 (C-3+C-4), 155.23(C-15), 172.12 (C-9);

MS (CI+): m/z=533.277 ([M]^(•+), 4.06), 478.591 ([MH—C₄H₈]^(•+), 2.61),434.267 ([MH—C₅H₈O₂]^(•+), 14.19), 91.129 ([C₇H₇]^(•+), 100.00);

HRMS calcd. for C₃₂H₃₉NO₆ ([M]^(•+), DCI, CH₄) 533.2777. found 533.2772.

Anal. calcd for C₃₂H₃₉NO₆: C, 72.02; H, 7.37; N, 2.62. Found: C, 71.48;H, 7.46; N, 2.17.

Compound 30 was obtained from Compound 28 as follows: To a solution ofCompound 28 (0.23 gram, 0.47 mmol) in 5.29 ml THF and 3.30 ml water wasadded 1 N LiOH solution (0.55 ml, 0.55 mmol) at 0° C. The solution colorturned immediately into yellow, and the solution was allowed to warm toroom temperature and stirred for 7.5 hours. Monitoring the reactionUsing TLC (hexane:EtOAc 2:1 v/v as eluent) indicated a presence of thestarting material Compound 28, and therefore additional 0.55 ml of 1 NLiOH solution were added, and the solution was further stirred at roomtemperature overnight. Upon reaction completion, as indicated by TLCanalysis, the solution was concentrated under vacuum to remove THF. Theresidue was acidified with 1 M KHSO₄ solution to obtain pH of about 1and the solution was extracted with EtOAc (×3). The organic layer waswashed with brine (×3), dried over Na₂SO₄, filtered and evaporated toafford Compound 30 as a white solid (0.17 gram, 76% yield).

Since the strongly basic medium could possibly give rise toepimerization at the α-carbon (resulting in racemization), the opticalactivity of Compound 30 was determined and found to be dextrorotatorywith specific rotation of [α]_(D) ²⁴=+12.0 (c 0.0057, MeOH), in goodaccordance with the value reported in literature.

In an alternative synthetic pathway, K₂CO₃ in H₂O—MeOH solution was usedas follows: K₂CO₃ (0.88 gram, 6.40 mmol) was added to a solution ofCompound 28 (0.90 gram, 1.83 mmol) in 28.73 ml MeOH and 3.93 ml water.The suspension was stirred at room temperature overnight. Water wasthereafter added, and the aqueous solution was acidified with 1 M KHSO₄solution until a pH of about 2 was obtained and was thereafter extractedwith DCM. The organic phase was washed with brine, dried over Na₂SO₄,filtered and evaporated to give Compound 30 as a white solid (0.85 gram,97% yield).

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.43 (s, 9H, H-14), 2.96 (dd,²J_(H-7,H-7′)=13.80 Hz, ³J_(H-7 or H-7′,H-8)=6.30 Hz, 1H, H-7 or H-7′),3.08 (dd, ²J_(H-7,H-7′)=13.80 Hz, ³J_(H-7 or H-7′,H-8)=4.80 Hz, 1H, H-7or H-7′), 4.55 (m [X of ABX system], 1H, H-8), 4.94 (br d, J=7.50 Hz,1H, H-11), 5.11 (two overlapping singlets, 4H, H-15+H-20), 6.69 (br dd,1H, H-6), 6.80 (br d, 1H, H-2), 6.85 (d, J_(ortho)=8.40 Hz, 1H, H-5),7.27-7.45 (m, 10H, H-17+H-18+H-19+H-22+H-23+H-24);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=28.43 (C-14), 37.35 (C-7), 54.41 (C-8),71.46 (C-15+C-20), 80.43 (C-13), 115.27 and 116.46 (C-2+C-5), 122.51(C-6), 127.41, 127.55, 127.89, 127.93, 128.58 and 128.60(C-17+C-18+C-19+C-22+C-23+C-24), 129.08 (C-1), 137.33 and 137.42(C-16+C-21), 148.27 and 149.00 (C-3+C-4), 155.54 (C-12), 176.19 (C-9);

The TEA salt of the obtained acid, Compound 30, was then alkylated withN-Boc-GABA-OCH₂Cl, Compound 23, in DMF, under heat, to afford theacyloxymethyl ester Compound 31 in 45% yield after purification by flashchromatography (see, Scheme 15 below).

Compound 23 was prepared from N-Boc-GABA as follows: To a solution ofN-Boc-GABA (1 equivalent) in water/DCM (1:1 v/v) were added NaHCO₃ (4equivalents), chloromethyl chlorosulfate (1.2 equivalent) andn-Bu₄N⁺HSO₄ ⁻ (catalytic amount). The mixture was stirred at roomtemperature for 18 hours. Subsequently, water was added to the mixture,the layers were separated and the aqueous phase was washed with DCM(×3). The combined organic layer was washed with 5% NaHCO₃ (×3) andbrine (×3), dried over Na₂SO₄, filtered and evaporated. The crudeproduct was purified by flash chromatography (using hexane:EtOAc 5:1 v/vas eluent, R_(f)=0.21) to afford Compound 23 as a colorless oil (59%yield).

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.45 (s, 9H, Me₃C), 1.86 (quint, J=7.05Hz, 2H, CH₂CH₂CH₂), 2.47 (t, J=7.35 Hz, 2H, CH₂CO₂), 3.18 (br t, J=6.75Hz, 2H, CH₂NH), 5.73 (br s, 1H, NH), 5.55 (s, 2H, OCH₂Cl);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=24.73 (CH₂CH₂CH₂), 28.13 (Me₃C), 30.93(CH₂CO₂), 39.39 (CH₂NH), 68.46 (OCH₂Cl), 78.81 (CMe₃), 155.92 (NHCO₂),171.12 (CO₂CH₂);

MS (CI+): m/z=252.102, 254.099 ([MH]^(•+), 33.13, 11.24), 196.019,198.019 ([MH—C₄H₈]^(•+), 100.00, 32.35), 152.005, 154.002([MH—C₅H₈O₂]^(•+), 54.07, 16.69), 130.052 ([C₆H₁₂NO₂]^(•+), 43.31);

HRMS calcd. for C₁₀H₁₉ClNO₄ ([MH]^(•+), DCI, CH₄) 252.1003, 254.0973.found

Conjugation of Compound 30 and Compound 23 to thereby obtain Compound 31was successfully performed as follows: To a solution of Compound 30 (124mg, 0.26 mmol) and Compound 23 (55 mg, 0.22 mmol) in DMF, under N₂atmosphere, was added TEA (0.1 ml, 0.72 mmol). The mixture was stirredand heated at about 70° C. for 4.5 hours. Upon reaction completion, asmonitored by TLC, as complete consumption of Compound 23, the solventwas evaporated to remove DMF, and the residue was taken up in EtOAc. Theorganic phase was washed with 5% NaHCO₃ (×3) and brine (×3), dried overNa₂SO₄, filtered and evaporated to give the crude product. Columnchromatography (using hexane:EtOAc 3:1 v/v as eluent) afforded Compound31 as a white solid (68 mg, 45% yield).

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.42 and 1.43 (two singlets, 18H,H-18+H-22), 1.79 (quint, J=6.98 Hz, 2H, H-13), 2.38 (t, J=7.20 Hz, 2H,H-12), 2.91-3.07 (m [AB of ABX system], 2H, H-7), 3.12 (br q, J=6.00 Hz,2H, H-14), 4.54 (br q, J=6.90 Hz, 1H, H-8), 4.72 (br s, 1H, H-15), 4.96(br d, J=6.60 Hz, 1H, H-19), 5.13 (s, 4H, H-23+H-28), 5.68 (d, J=5.40Hz, 1H, H-10), 5.75 (d, J=5.40 Hz, 1H, H-10′), 6.66 (dd, J_(ortho)=8.10Hz, J_(meta)=1.80 Hz, 1H, H-6), 6.76 (d, J_(meta)=1.80 Hz, 1H, H-2),6.86 (d, J_(ortho)=8.10 Hz, 1H, H-5), 7.28-7.45 (m, 10H,H-25+H-26+H-27+H-30+H-31+H-32);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=25.07 (C-13), 28.40 and 28.50(C-18+C-22), 31.17 (C-12), 37.34 (C-7), 39.71 (C-14), 54.37 (C-8), 71.43and 71.53 (C-23+C-28), 79.69 (C-10), 80.256 (C-17+C-21), 115.30 and116.47 (C-2+C-5), 122.49 (C-6), 127.39, 127.48, 127.91, 127.96, 128.59and 128.59 (two overlapping signals, C-25+C-26+C-27+C-30+C-31+C-32),128.91 (C-1), 137.26 and 137.35 (C-24+C-29), 148.36 and 149.06(C-3+C-4), 155.19 and 156.08 (C-16+C-20), 170.94 and 171.83 (C-9+C-11);

MS (CI+): m/z=692.331 ([M]^(•+), 32.03), 593.419 ([MH—C₅H₈O₂]^(•+),100.00), 537.385 ([MH—C₅H₈O₂—C₄H₈]^(•+), 53.81), 493.369([MH—C₅H₈O₂—C₅H₈O₂]^(•+), 20.75);

HRMS calcd. for C₃₈H₄₈N₂O₁₀ ([M]^(•+), DCI, CH₄) 692.3309. found692.3315.

The final stage required removal of the hydroxy and amino protectinggroups, as depicted in Scheme 16 below.

Hydrogenolysis using 10% Pd catalyst in a Parr shaker apparatus at 50psi and room temperature furnished the free Compound 32 within 2.5hours. Thus, hydrogenolysis was successfully performed as follows: To asolution of Compound 31 (99 mg, 0.14 mmol) in MeOH was added 10% Pd/C(10 mg). The suspension was then hydrogenated in a Parr apparatus under50 psi of H₂ for 2.5 hours. The obtained mixture was filtered andevaporated, to give the desired product as a yellowish oil (60 mg, 82%yield). The compound was used in the next step without furtherpurification.

¹H NMR (300 MHz, CDCl₃) ppm: δ=1.42 and 1.43 (two singlets, 18H,H-18+H-22), 1.79 (quint, J=6.98 Hz, 2H, H-13), 2.38 (t, J=7.20 Hz, 2H,H-12), 2.91-3.07 (m [AB of ABX system], 2H, H-7), 3.12 (br q, J=6.00 Hz,2H, H-14), 4.54 (br q, J=6.90 Hz, 1H, H-8), 4.72 (br s, 1H, H-15), 4.96(br d, J=6.60 Hz, 1H, H-19), 5.13 (s, 4H, H-23+H-28), 5.68 (d, J=5.40Hz, 1H, H-10), 5.75 (d, J=5.40 Hz, 1H, H-10′), 6.66 (dd, J_(ortho)=8.10Hz, J_(meta)=1.80 Hz, 1H, H-6), 6.76 (d, J_(meta)=1.80 Hz, 1H, H-2),6.86 (d, J_(ortho)=8.10 Hz, 1H, H-5), 7.28-7.45 (m, 10H,H-25+H-26+H-27+H-30+H-31+H-32);

¹³C NMR (75 MHz, CDCl₃) ppm: δ=25.02 (C-13), 28.39 and 28.50(C-18+C-22), 31.15 (C-12), 37.40 (C-7), 39.89 (C-14), 54.44 (C-8)¹,79.64 (C-10), 80.14 and 80.50 (C-17+C-21), 115.42 and 116.38 (C-2+C-5),121.44 (C-6), 127.50 (C-1), 14²3.56 and 144.46 (C-3+C-4), 155.46 and156.72 (C-16+C-20), 171.12 and 171.92 (C-9+C-11).

The two N-Boc moieties were deprotected as follows: To an ice-coldsolution of Compound 32 in EtOAc a freshly prepared solution of 4 N HClin EtOAc was added (obtained by addition of a known amount of acetylchloride to an ice-cold solution of an equivalent amount of EtOH inEtOAc). The ice bath was removed after 1 hour, and the solution wasallowed to warm to room temperature. After TLC analysis had showncomplete consumption of the Boc-protected Compound 32, the solvent wasevaporated to give the desired Compound 33 as a dihydrochloride salt ina quantitative yield.

¹H NMR (200 MHz, CD₃OD) ppm: δ=0.91 (t, 3H, J=7.20 Hz, H-13), 1.32 (m,2H, H-12), 1.62 (m, 2H, H-11), 1.97 (quint, J=7.50 Hz, 2H, H-13), 2.57(t, J=7.10 Hz, 2H, H-12), 3.01 (t, J=7.70 Hz, 2H, H-14), 3.09 (d, J=6.40Hz, 2H, H-7), 4.32 (t, J=6.60 Hz, 1H, H-8), 5.80 (d, J=5.80 Hz, 1H,H-10), 5.89 (d, J=5.80 Hz, 1H, H-10′), 6.59 (dd, J_(ortho)=8.00 Hz,J_(meta)=2.00 Hz, 1H, H-6), 6.70 (d, J_(meta)=2.00 Hz, 1H, H-2), 6.76(d, J_(ortho)=8.00 Hz, 1H, H-5);

¹³C NMR (50 MHz, CD₃OD) ppm: δ=23.27 (C-13), 31.17 (C-12), 36.50 (C-7),39.86 (C-14), 55.11 (C-8), 81.21 (C-10), 116.79 and 117.40 (C-2+C-5),121.94 (C-6), 125.93 (C-1), 146.07 and 146.65 (C-3+C-4), 169.16 and172.36 (C-9+C-11).

Example 2 Effect of L-DOPA-GABA Conjugate (BL-1023; Compound 5) onMPTP-Induced Parkinson's Disease Model in Mice

Parkinson's disease (PD) is a neurodegenerative disorder characterizedby reduction in striatal dopamine (DA) content caused by the loss ofdopaminergic neurons in the Substantia Nigra pars compacta (SNpc) andtheir projections to the striatum. MPTP is an effective dopaminergicneurotoxin in mouse. MPTP administration lead to bilateral marked lossof Tyrosin-Hydroxilase (TH) immunoreactive cell bodies in the SNpc.

Materials:

BL-1023 conjugate was synthesized as described in Example 1 hereinaboveand was dissolved in saline to achieve equimolar doses of 20 mg/kg and40 mg/kg L-DOPA. The solutions were freshly prepared prior toadministration.

L-DOPA (Sigma Cat. No. D9628) was dissolved in saline to achieve aconcentration of doses of 20 mg/kg and 40 mg/kg. The doses were freshlyprepared prior to administration.

Solution of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride(MPTP; Sigma (Cat. No. M0896) was freshly prepared prior to eachinjection session by dissolving the lyophilized powder in saline toachieve a final injected concentration of 4 mg/ml, appropriate for theselected dose of 20 mg/kg and dose volume of 5 ml/kg.

Rabbit antibody against Tyrosine Hydroxylase (TH) was purchased fromCalbiochem and was stored at −80° C. following receipt until being used.

Gout anti Rabbit antibody was purchased from Pharmatraide and was storedat −80° C. following receipt until being used.

Assay Protocol:

Young adult (7-8 weeks old) male mice strain C57/bl were acclimated forone week prior to MPTP initiation. During acclimation and throughout theentire study duration, animals were housed within a limited accessrodent facility and kept in groups of maximum 5 mice in polypropylenecages (23×17×14 cm), fitted with solid bottoms and filled with woodshavings as bedding material. Animals were provided ad libitum acommercial rodent diet and free access to drinking water, supplied toeach cage via polyethylene bottles with stainless steel sipper tubes.

Automatically controlled environmental conditions were set to maintaintemperature at 20-24° C. with a relative humidity (RH) of 30-70%, a12:12 hour light:dark cycle and 15-30 air changes per hour in the studyroom.

Animals were given a unique animal identification tail mark. This numberalso appears on a cage card, visible on the front of each cage. The cagecard also contained the study and group numbers, route ofadministration, gender, strain and all other details relevant to thetreatment group. During the acclimation period, animals were randomlyassigned to experimental groups.

Mice were administered IP with 4 injections of MPTP (each injectioncontained 20 mg/kg, 5 ml/kg) in saline or saline alone (control) at 2hours interval on day 0. Mice were then grouped as described in Table 3below and were administered subcutaneously (cuss.) with test solutionsby once daily repeated dosing sessions throughout 8 successive treatmentdays (Days 0-7), as illustrated in FIG. 1.

TABLE 3 Volume Test Group Dose Level dosage Material size Route(mg/kg/admin) (ml/kg) Regime Naïve N = 3 Vehicle n = 10 S.C 0 20 Oncedaily control from day 0 Positive n = 10 S.C 40 mg/kg 20 Once dailyControl from day 0 L-DOPA (40 mg/kg) BL-1023 n = 10 S.C equimolar to 4020 Once daily equimolar mg/kg L-DOPA from day 0 to 40 mg/kg

Seven days following MPTP administration animals are euthanized andtheir brains were removed for immunohistochemistry analysis of IR THcells at the level of the SNpc.

Brains are fixed by cardiac perfusion with 4% Paraformaldehyde followedby fixation by immersion with the same fixative for at least 72 hours.Brains were then washed with PBS and transferred to 30% sucrose in PBSuntil they sank. Brains were then frozen using the craryostat specialfast freezing (−60° C.). Brains were then crayosectioned (20 μm) at thelevel of the striatum and at the level of the substantia nigra (SN).

Immunohistochemistry staining was performed using Rabbit anti tyrosinhydroxylase (1:100). Slides were stained using DAB detection kit(Pharmatraide). Quantitative analysis was effected by counting ofimmunoreactive cells at the widest dimension of the SNpc lateral to theroots of the third cranial nerve separating medial and lateral SNpc atthe level of interpreduncular nucleus.

Results:

As can be seen in Table 4 below, the number of TH immunoreactive cellbodies in the Substantia Nigra pars compacta (SNpc) of mice which weretreated with BL-1023, was 41.0% higher than in the SNpc of mice treatedwith L-DOPA and 77.4% higher than in the SNpc of vehicle treated(control) mice.

Without being bound to any particular theory, it is suggested that thehigher efficiency of BL-1023, as compared with an equimolar dose ofL-DOPA, may be attributed to higher amount of available dopamine in theSNpc. Since L-DOPA is partially metabolized to dopamine in peripheraltissues by aromatic-L-amino-acid decarboxylase, and since dopamine perse is incapable of crossing the blood-brain barrier (BBB), the amount ofdopamine eventually reaching the SNpc is substantially diminished. Onthe other hand, since BL-1023 is likely not metabolized byaromatic-L-amino-acid decarboxylase until after crossing the BBB, theamount of dopamine reaching the SNpc via systemic administration ofBL-1023 is substantially higher.

Hence, these results clearly indicate that systemically administeredBL-1023 is substantially more effective than L-DOPA in protectingagainst the loss of dopaminergic neurons in the Substantia Nigra parscompacta of Parkinson's model animals.

TABLE 4 Number of TH Standard Treatment Cells Error Naïve 101 8 Vehiclecontrol 31 16 L-DOPA (40 mg/kg) 39 10 BL-1023 (equimolar 55 4 to 40mg/kg L-DOPA)

Example 3 Effect of L-DOPA-GABA Conjugate (BL-1023* and AN-490) onMPTP-Induced Parkinson's Disease Model in Mice

The protective effect of L-DOPA-GABA conjugates BL-1023* and AN-490 wastested using the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)mice model.

Materials and Methods:

Male C57/bl mice were obtained from Harlan, Israel and were housed incontrolled conditions for one week prior to the experiments. All animalexperiments were conducted according to the NIH Laboratory Animal CareGuidelines and with the approval of the Tel Aviv University Committeefor Animal Experimentation (permit M-08-073).

Mice Model of MPTP Induced Parkinson:

Acute model: On day 1, C57/BL mice received 4× subcutaneous injectionsof 20 mg/kg MPTP-HCl (Sigma), dissolved in saline solution, at 2 hoursintervals.

Subacute model: Once a day, for five consecutive days, the animalsreceived a subcutaneous injection of 20 mg/kg MPTP-HCl.

Tests Used to Monitor the Treatment Effects:

Motor behavior: The effect of the treatment on motor behavior wasassessed in the RotoRod and open-field tests, as follows.

RotoRod Test: The test measures and records motor coordination of ratsand mice using the natural fear of falling motivation. RotoRod (SanDiego Industries, San Diego, Calif., USA) was set on 16 rpm speed andaccelerated to maximum speed of 25 rpm. The time until the mouse fallwas measured up to maximum 4 minutes. The measurements were performedthree times for each mouse and the average was calculated. Four animalswere placed on the rod on individual lanes in the RotoRod enclosure.Photo beams were embedded in each of the four lanes of the enclosure.When the animal falls from the rotating rod, the photo beams were brokenand the Rota-Rod recorded the animal's latency to fall. When the photobeams in all four lanes have been broken, the rod stopped rotating.

The Open Field Test:

The open field test measures behavioral responses, locomotor activity,hyperactivity, and exploratory behaviors. Open field test is also usedas a measure of anxiety. Mice tend to avoid brightly illuminated, novel,open spaces, so the open field environment acts as an anxiogenicstimulus and allows for measurement of anxiety-induced locomotoractivity and exploratory behaviors. The apparatus for the open fieldtest is a square (0.5×0.5 meter) made of white melamine. All activitieswere recorded by a video camera mounted above the open field and scoredin real-time by a motion-recognition software package (Noldus, Holland)that detects and analyzes the movements of the animals. The video imageof the open field arena was partitioned into equal-size squares zone.Total distance, average speed, rearing/elongation behavior, and timespent in various parts of the field (e.g. the border areas vs. mid-area)were measured. Testing was carried out in a temperature, noise and lightcontrolled room.

Tyrosine Hydroxylase Immunohistochemistry:

Animals under deep anesthesia were perfused through the aorta withphosphate-buffered saline (PBS), followed by a cold fixative consistingof 4% paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acid inphosphate buffer. After perfusion, the brain were removed and fixed in4% paraformaldehyde for 24 hours, washed with PBS, dehydrated inincreasing alcohol concentrations and embedded in paraffin.Paraffin-embedded tissues were processed as described [Rephaeli et al.Int J Cancer. 2005 Aug. 20; 116:226-35]. The avidin-biotin nonspecificbinding was prevented by a blocking kit according to the manufacturer'sprotocol (Vector, USA). The sections were further incubated at 4° C.overnight with the primary antibody mouse monoclonal tyrosinehydroxylase (Visionbiosystem, Newcastle, UK, diluted 1:50). Thesecondary antibody was biotin conjugated goat anti-mouse IgG (SantaCruz). Slides were then stained with ABC peroxidase system, developedwith diaminobenzidine (DAB), chromogene substrate (Vector Laboratories,Inc. The slides were examined using an Olympus BX52 light microscope andimages were taken with Olympus DP50 digital camera system. Images wereanalyzed by the ImagePro Plus 5.1 software. At least 12 different fieldsin each experimental group were analyzed. The Mean density±SEM wascalculated for the striatum and the mean cell number±SEM was calculatedfor the SN.

Measurement of Dopamine and Catecholamines Content in the Brain:

The mice were killed by decapitation. Brains were rapidly removed andimmediately frozen in liquid nitrogen, and stored at −80° C. untilanalyzed. For the assay, the thawed brain was homogenized on ice in 2 mlof 0.2 M perchloric acid. After centrifugation (10 000×g for 15 minutesat 4° C.), the supernatant was filtered and centrifuged throughpolypropylene Spin-X® centrifuge tube filter 0.22 μm nylon (Costar,Corning, N.Y., USA). Determination of the level of catecholamines undergood laboratory practice was performed in the chemical laboratory ofRabin medical center. An aliquot of filtrate was injected into the HPLCsystem (Waters, Milford, Mass., USA) equipped with a C18 reverse phase,3-μ LUNA column (100 mm×2 mm, Phenomenex, Torrance, Calif., USA).High-performance liquid chromatography (HPLC) with electrochemicaldetection was used to measure dopamine. Results were validated byco-elution with CA standards under various buffer conditions anddetector settings. Results were validated using catecholamines(dopamine, L-DOPA, norepinephrine) standards. The samples of the Naïveand MPTP control groups were pooled from Experiments 1 and 3, asdescribed hereinbelow.

Experiment 1: Protective Effect of AN-490 HCl Salt (Compound 21) in MPTPAcute Model

-   Mice: 60 Males C57/BL/6J mice, 11 weeks old, weighing 22-25.5 grams.-   All compounds were dissolved in saline. The concentration of the    AN-490 compound was not corrected for purity.-   MPTP administration: 4 IP injections of MPTP (Sigma)×20 mg/kg every    2 hours on day 0.

AN-490, as an HCl salt (shown below), was synthesized as described inExample 1 hereinabove and stored in desiccator in the cold room, and wasbrought to room temperature prior to weighing and dissolving it insaline. The volume of administration was 10 ml/kg.

-   Treatment groups (6 mice per group):    -   Group 1—Control, saline treated mice    -   Group 2—L-DOPA 25 mg/kg (per os)    -   Group 3—AN-490 67.5 mg/kg (equimolar dose to 25 mg/kg L-DOPA)    -   Group 4—MPTP only    -   Group 5—MPTP+L-DOPA    -   Group 6—MPTP+AN-490 67.5 mg/kg-   Treatment schedule: Days: 0, 1, 2, 3, 6, 7, 13, 16 by oral gavage.-   Open field: was performed on days: 3, 6, 8, 16 (performed 90 minutes    after treatment). Distance moved, velocity, immobility, strong    mobility were measured.-   Rota-Rod: was performed on days: 1, 3, 6 (performed 180 minutes    after treatment)-   Catecholamines biochemistry: Brains for dopamine were taken on day    20 and kept frozen in −70° C.

Experiment 2: Protective Effect of AN-490 HCl Salt (Compound 21) in MPTPSub-Acute Model

-   Mice: 60 C57/BL/6J mice (Harlan, Israel), 9-12 weeks old weighing    22-25.5 grams.-   MPTP administration: Once a day, IP injection of 20 mg/kg for 5    consecutive days.-   Treatment groups: 6 groups (10 mice per group):    -   Group 1: Control, vehicle treated mice    -   Group 2: MPTP+saline per os    -   Group 3: MPTP+GABA.HCl 18.4 mg/kg (equimolar dose to 30 mg/kg        L-DOPA) per os    -   Group 4: MPTP+L-DOPA 30 mg/kg per os    -   Group 5: MPTP+L-DOPA 30 mg/kg+GABA 18.4 mg/kg per os    -   Group 6: MPTP+AN-490 81 mg/kg per os-   Treatment schedule: Starting from day 13, every day for 6 days and    in the second week, due to observational signs of toxicity, the    treatment was reduced to three times a week.-   Behavioral studies: Open field test was performed 90 minutes prior    to treatment, on days 11, 20 and 27 (during treatment) and Rota-Rod    test was performed on day 24.-   Immunohystochemistry (IHC): At termination, on days 29-30 of the    treatment, the mice were perfused and the brains were taken for IHC    staining for tyrosine hydroxylase.

Experiment 3: Protective Effect of BL-1023* (Compound 16) in MPTP AcuteModel

BL-1023*, as an HCl salt (shown below) was prepared as described inExample 1 hereinabove. MW: 318.75 grams/mol. Appearance: brown powder.Purity: 90.12%. The concentration of the BL-1023* compound was notcorrected for purity.

-   Mice: 60 C57/BL/6J (Harlan, Israel) 9 weeks old weighing 22-25.5    grams.-   MPTP administration: 4 imp injections of MPTP×20 mg/kg every 2 hours    on day 1.-   Treatment groups: 6 groups (10 mice per group):    -   Group 1: Control, saline treated mice    -   Group 2: MPTP+saline per os    -   Group 3: MPTP+L-DOPA (30 mg/kg L-DOPA) per os    -   Group 4: MPTP+GABA 18.4 mg/kg (equimolar)+L-DOPA (30 mg/kg per        os)    -   Group 5: MPTP+BL-1023* 48.4 mg/kg (equimolar to 30 mg/kg L-DOPA)        per os    -   Group 6: MPTP+BL-1023* 24.2 mg/kg (equimolar to 15 mg/kg L-DOPA)        per os-   Treatment schedule: Starting at day 7 and then after 9, 11 and 13    days (total of 4 treatments)-   Behavioral studies: Open field—day 13.    -   Rotarod—on day 6 prior to treatment    -   Rotarod—on day 12 (after two treatments).-   Immunohystochemistry and catecholamine concentrations: Samples for    IHC (3 mice per group) and for determination of catecholamines (3    mice per group) were taken on days 14-15.    Results:

Experiment 1: AN490-Acute Model

Four parameters of mice behavior in the open field paradigm werefollowed: distance moved, velocity, immobility and strong mobility. Theresults are presented in FIGS. 2A-2H.

In mice that were not treated with MPTP, L-DOPA (25 mg/kg per os)induced a tendency toward increase in motility compared to naïve mice ormice treated with equimolar dose of AN-490. This was evident by increasein distance moved, velocity and strong mobility, and a decrease in thetime spent immobile behavior (see, FIG. 2). MPTP and L-DOPA treatmentlower the motility of the mice Compared to all other treated groups andcontrol naïve mice. The immobility and strong mobility of MPTP+L-DOPAvs. MPTP+AN-490 was significant (p<0.05). Interestingly, it can be seenthat MPTP as well as MPTP+L-DOPA treatments induced an initial increase(day 3-6) in motility. On day 8, decreased motility and recovery on day16 were observed. In the MPTP+AN-490 group, a continuous increase inmotor activity was noted during the period of the study. Since it islikely that the MPTP treatment collapsed the BBB, GABA released byAN-490 evidently reaches the brain and imparted its protective activity.

It is important to mention that the test was not full established sincenone of the tested parameters was significantly reduced in the MPTPgroup relatively to the naïve group.

Consistent with the open field observations, it was found that in theRotarod all the MPTP groups showed a tendency towards shorter latencytime (see, FIG. 3).

The data obtained for changes in body weight as a parameter of toxicityshow that the lowest average body weight was of the mice treated withMPTP+L-DOPA (see, FIG. 4).

In the study described herein the mice received MPTP and at the same dayand thereafter they also were given the other treatments. This scheduleof treatment is not reported in the literature. In most studies afterthe administration of MPTP the mice allow to recover for a week or more.The simultaneous treatment could account for the toxicity observed withL-DOPA.

Experiment 2: AN490-MPTP Sub-Acute Model

In this study the effect of treatments with AN-490, L-DOPA, GABA andL-DOPA+GABA on mice treated subchronically with MPTP was evaluated. Theresults on the Rotarod performed on day 24 and the behavior parametersin the open field test performed on days 11, 20 and 27 of the study,show no differences among the treated groups and the placebo animals(see, FIG. 5). AN-490 treated mice showed reduced vitality signs (furappearance; see, Table 5 and FIG. 7), which was presumably attributed tohydrolysis of the conjugate.

As shown in FIG. 8, the IHC staining of brain demonstrated thedisappearance of TH positively staining in the substantia nigra andstriatum in mice treated with MPTP and protection or restoration of thenormal morphology by GABA, GABA and L-DOPA and AN-490.

TABLE 5 Observation Observed parameters observer 1 observer 2 observer 3observer 4 Vital- Vital- Vital- Vital- Group ity Fur ity Fur ity Fur ityFur Control +++ +++ +++ +++ +++ +++ +++ +++ MPTP ++ +++ ++ +++ +++ +++++ +++ MPTP + +++ +++ +++ ++ +++ +++ +++ +++ GABA MPTP + ++ +++ ++ ++++++ +++ +++ +++ L-DOPA MPTP + ++ +++ ++ ++ +++ +++ +++ +++ GABA + DopaMPTP + +++ + +++ + +++ ++ +++ ++ AN-490 Split fur + Shiny fur +++ slowmotions + normal motion +++

These data suggest that MPTP could interfere with the intactness of theBBB, thus allowing the penetration of GABA to the brain, where itexhibits a neuroprotective effect. These data further show that AN-490also exhibits TH protective activity, which could possibly be due to therelease of GABA.

Experiment 3: MPTP Acute Model with BL-1023*

Behavioral parameters: In this experiment a protective effect of BL1023*against the shorter latency in the rotarod test, induced by MPTPtreatment, was observed. The results are presented in FIG. 9, and showthat this latency was not attenuated by L-DOPA or by the combination ofL-DOPA and GABA. The behavioral signal was found in the rotarod, but notin the open field (see, FIG. 10), supporting the view that the openfield test is not sufficiently indicative of anti-Parkinson activity inthis model.

The level of catecholamines: As shown in FIG. 12, the level of dopaminein the brains of all MPTP treated mice was significantly lower than thelevel in the brains of naïve mice, regardless of the treatment used. Inall MPTP treated mice the level of L-DOPA was higher than in the naïvemice, suggesting that MPTP blocks the synthesis of catecholamines afterTH.

The level of norepinephrine in the MPTP treated group was significantlylower than the naïve group. The level of norepinephrine in MPTP+L-DOPAor in MPTP+L-DOPA and GABA treated mice was similar to that of naïvemice, suggesting that treatment with L-DOPA results in a higher level ofthis catecholamine. Treatment with BL-1023* did not restore thenorepinephrine levels.

Staining for TH in the striatum and the substantia nigra: Along with theprotective effect of BL1023* in the rotarod, IHC staining for TH in thestriatum, exhibited a marked neuroprotective effect, expressed by asignificant higher TH levels in the BL-1023* treated animals as comparedto MPTP or MPTP combined with L-DOPA or with L-DOPA and GABA (see, FIGS.13-16). Overall it can be concluded that BL-1023* and GABA protect theTH positive cells.

Example 4 Specific Binding of BL-1023 (Compound 5) to Various Proteinsand Receptors Using Radioligand Binding Assay

The binding of BL-1023 to the following proteins/receptors was assessed:Adenosine A₁, Adenosine A_(2A,) Adenosine A_(3,) Adrenergic α_(1.)Non-Selective, Adrenergic α_(2.) Non-Selective, Adrenergic β₁,Adrenergic β₂, Angiotensin AT₁, Bradykinin B₂, Chemokine CCR1, ChemokineCXCR2 (IL-8R_(B)), Dopamine D₁, Dopamine D_(2L), Dopamine D_(2S),Dopamine D₃, Dopamine D_(4.2), Dopamine D_(4.4), Dopamine D_(4.7),Dopamine D₅, Endothelin ET_(A.) Endothelin ET_(B.) GABA_(A)-ChlorideChannel-TBOB, GABA_(A), -Chloride Channel-TBPS,GABA_(A)-Muscimol-Central, GABA_(A)-Ro-15-1788-Cerebellum,GABA_(A)-Ro-15-1788-Hippocampus, GABA_(B1A), GABA_(B1B), Histamine H1,Histamine H2, Histamine H3, Muscarinic M1, Muscarinic M2, Muscrinic M3,Neuropeptide YY1, Neuropeptide YY2, Neurotensin NT1, Opiate δ (OP1,DOP), Opiate κ (OP2, KOP), Opiate μ (OP3, MOP), Serotonin 5-HT_(1A),Serotonin 5-HT_(1B), Serotonin 5-HT_(2A), Serotonin 5-HT_(2B), Serotonin5-HT₃, Serotonin 5-HT_(5A), Serotonin 5-HT₆, Serotonin 5-HT₇, Sigma σ₁,Sigma σ_(2.) Sodium channel, site 2, Somatostatin sst1, Somatostatinsst2, Somatostatin sst3, Somatostatin sst4, Somatostatin sst5,Tachykinin NK1, Tachykinin NK2, Dopamine Transporter (DAT),Norepinephrin Transporter (NET), Vasoactive Intestinal Peptide (VIP₁),and Vasopressin V_(1A).

Radioligand Assay:

The binding of BL-1023 to the various proteins/receptors was determinedusing a radioligand assay. In the radioligand assay the binding affinityof BL-1023 to a specific receptor/protein target was determined byassessing the percent of inhibition of the binding of a well-knownradiolabeled ligand to the specific protein/receptor target in thepresence of BL-1023. Reference standards were run as an integral part ofeach assay to ensure the validity of the results obtained.

The IC50 values were determined by a non-linear, least squaresregression analysis using MathIQ™ (ID Business Solutions Ltd., UK) andinhibition constants (Ki) were calculated using the equation of Chengand Prusoff (Cheng, Y., Prusoff, W. H., Biochem. Pharmacol.22:3099-3108, 1973) using the observed IC50 of the tested compound, theconcentration of radioligand employed in the assay, and the historicalvalues for the K_(D) of the ligand.

The significance criteria were a>50% stimulation or inhibition of theradioligand binding to its target protein/receptor.

Results:

BL-1023, at concentrations up to 10 μM, was not found to significantlyinhibit/stimulate the binding of any of the radioligands to the testedproteins/receptors. These results therefore suggest that the extent ofbinding of BL-1023 to the tested protein/receptors is relatively low.

Example 5 Therapeutic Potential of BL-1023 (Compound 5) AfterDevelopment of MPTP Lesion: Up Scaled Study

The study assess the potential of BL-1023 to correct locomotor deficits,restore DA levels in the striata, and induce TH sprouting of neuronsafter lesion development. For these studies mice are first intoxicatedwith acute doses of MPTP (4 injections of 18 mg/kg, IP) which inducesstrong microglial inflammatory responses with virtual complete loss ofstriatal TH termini and robust nigral neurodegeneration withapproximates 70%. The study is developed into 2 parts:

Part 1. Mice are treated with BL-1023 and L-DOPA 24 hours prior to and12 hours following MPTP intoxication. Daily administration continuesuntil day 6, in order to allow for metabolism and excretion of themajority of the MPTP and MPP+. This staggered initiation precludes thepossibility that excess dopamine either from BL-1023 or L-DOPA mayinhibit MPP+ uptake by neuronal dopamine transporters. The intent is togive the drug before and at the time the lesion is developing to exertthe therapeutic effect for that time. The purpose of giving the drug at12 hours post MPTP is to initiate drug as soon as possible after MPTPwithout the drug interfering with the metabolism of MPTP to MPP+ andlimiting inhibition of MPP+ uptake by DA neurons via competition withexcess drug-derived dopamine in the brain. Computerized spreadsheetscontaining mouse ear tag number, date of birth, date of death orsacrifice, dose and treatment schedule, body weight and behavioral dataare maintained. All animal procedures follow the guidelines establishedby the National Institutes of Health guidelines and be approved by theInstitutional Animal Care and Use Committee of the University ofNebraska Medical Center.

Part 2. At day 6 post MPTP treatment, one set of mice are sacrificed andthe extent of MPTP lesion and DA loss is assessed. Drug treatment(L-DOPA and BL-1023) is administered daily from day 7 and proceed untilday 35 (28 days post-MPTP). During that time, mice are assessed forlocomotor function by overall rotarod performance and grip strength. Ondays 14, 21, 28 and 35, striatal catecholamine levels and the extent ofTH sprouting by any remaining dopaminergic neurons is assessed. Inaddition, mice are monitored for rotarod performance twice every weekand are pre-conditioned for 3 days prior to testing. Mice are placed ona partitioned rotating rod (Rotamex Rota-rod apparatus, ColumbusInstruments, Columbus, Ohio) and tested at a 5 10, and 15 rpm for amaximum of 90 seconds at each speed with a minimum of 5 minutes restbetween attempts. The overall rotarod performance (ORP) is calculated asthe area under the curve using Prism (version 4, Graphpad Software, SanDiego, Calif.) from the plot of the time that the animal remained on therod as a function of the rotation speed. Grip strength of hind limbs ofmice is assessed each week. Each mouse is placed on the wire-lid of aconventional housing cage and gently shaken to prompt the mouse to holdon to the grid. The lid is turned upside down and the durationdetermined until the mouse released both hind limbs. Each mouse is giventhree attempts with a maximum duration of 90 seconds and the longestlatency is recorded. All animals are assessed every 3 days for bodyweight and weekly for signs of motor deficit: 4 points if normal (nosign of motor dysfunction), 3 points if hind limb weakness is evidentwhen suspended by the tail, 2 points if gait abnormalities are present,and 1 point for dragging of at least one hind limb.

Mice were divided to the following treatment groups (20-60 mice pergroup):

-   1 PBS Control-   1 MPTP Control-   3 40 mg/kg BL-1023 in the post-MPTP treatment schedule: administered    24 hours before MPTP intoxication and12 hours post MPTP    intoxication. Administration continues until day 6.-   4 24.8 mg/kg L-DOPA in the post-MPTP treatment schedule:    administered 24 hours before MPTP intoxication and12 hours post MPTP    intoxication. Administration continues until day 6.-   5 40 mg/kg BL-1023 in the post-MPTP treatment schedule: administered    6 days post MPTP intoxication for 28 days.-   6 24.8 mg/kg L-DOPA in the post-MPTP treatment schedule:    administered 6 days post MPTP intoxication for 28 days.

Material and Methods:

Animals:

Male 6-7 week old, WT C57BU6J (stock 000664) are purchased from JacksonLaboratories (Bar Harbor, Me.). All animal procedures are performed inaccordance with National Institutes of Health (NIH) guidelines andapproved by the Institutional Animal Care and Use Committee (IACUC) ofthe University of Nebraska Medical Center (UNMC).

Drug Treatments:

Mice are administered daily IP doses of BL-1023 (40 mg/kg) or L-DOPA(24.8 mg/kg, the equivalent amount of L-DOPA contained in BL-1023)beginning one day before the MPTP intoxication (groups 3 and 4) and 7days after MPTP intoxication (groups 5 and 6). Daily administration ofthe drug during the 6 days (groups 3 and 4) is to maintain drug duringlesion development.

The effect of drug given during the 28 days after lesion development(groups 5 and 6) is also examined as well as the therapeutic efficacy ofthe drug after lesion development by testing recovery of locomotionduring that time and dopaminergic termini sprouting at the end of thatperiod (day 35 post MPTP).

MPTP Intoxication:

For acute intoxications, mice receive 4 subcutaneous injections, oneinjection every 2 hours, of either vehicle (PBS, 10 ml/kg) orMPTP-HCl/PBS (Sigma-Aldrich, St. Louis, Mo.) [18 mg/kg in 10 ml/kg,based on free base]. MPTP handling and safety measures are performed inaccordance with published guidelines.

At appropriate time points following MPTP intoxication, mice areterminally anesthetized and transcardially perfused with 4%paraformaldehyde (PFA) in 0.1 M PBS using 0.9% saline as vascular rinse.The sacrificing is done on the same group of animals who undergo thebehavioral tests. Brains are post-fixed in 4% PFA overnight, kept in 30%sucrose for 2 days, snap frozen, embedded in O.C.T compound, and 30 μmsections cut with a cryostat (CM1900, Leica, Bannockburn, Ill.). Thesections are collected in PBS with sodium azide and processedfree-floating for staining of striatal and nigral TH expression bydopaminergic neurons and MAC-1 expression by nigral microglia. Primaryantibodies for immunohistochemistry include rabbit anti-TH antibody(1:2000; Calbiochem/EMD Biosciences, Inc., San Diego, Calif.) and ratanti-mouse CD11b or MAC-1 (1:1,000; Serotec, Raleigh, N.C.).Immunostaining is visualized in the substantia nigra and striatum usingdiaminobenzidine (Sigma-Aldrich) as the chromogen and mounted on slides.Immunostained brain sections is then counterstained with thionin(Sigma-Aldrich). Fluoro-Jade C (Chemicon International, Inc., Temecula,Calif.) is used to stain degenerating neurons in substantia nigra afterday 2 post MPTP and is detected as green fluorescence by fluorescencemicroscopy with FITC filter (Eclipse E800, Nikon, Inc., Melville, N.Y.).To assess reactive microglia, midbrain sections (30 μm) from 5-7 miceper treatment group (12 sections per animal), numbers of amoeboid MAC-1⁺cells per mm² is obtained within the SN and averaged for each animal.

Measurement of Striatal Catecholamines and TH-Positive Neurons andTermini:

Striatal dopamine and its metabolites, dihydroxyphenylacetic acid, andhomovanillic acid (HVA), is analyzed 6, 14, 21, 28 and 35 days afterMPTP intoxication by reverse-phase HPLC using electrochemical detection.Briefly, striata are harvested and sonicated in 50 volumes (w/v) 0.1 Mperchloric acid/10⁻⁷ M ascorbic acid containing 50 ng/mldihydrobenzylamine as internal standard. After centrifugation at 15,000g for 15 minutes at 4° C., 20 μl of supernatant is injected onto aC18-reverse-phase HR-80 catecholamine column (ESA, Bedford, Mass.) at25° C. The mobile phase consists of 90% 50 mM sodium phosphate/0.2 mMEDTA/1.2 mM heptanesulfonic acid (pH 3.2) solution and 10% methanol andis pumped at 1.0 ml per minute. Peaks are detected at +750 mA using anelectrochemical detector (BAS, West Lafayette, Ind.) with a glassycarbon working electrode and a Ag/AgCl reference electrode; peak area iscompared to the internal standard peak area. Data are collected andprocessed using the EZ Start data analysis software (ShimadzuScientific, Columbia, Md.). Catecholamine levels are quantitated bycomparison of peak areas to those of known standards of variousconcentrations spiked into control matrix. Stock solutions ofcatecholamines are made in 100% methanol at a concentration of 1.0 mg/mland stored for up to 3 months at −20° C. Catecholamines are weighed on aFisher Scientific accu-124 analytical balance (verified with referenceweights) and an appropriate amount of HPLC-grade methanol is added tothe measured standard to achieve a concentration of 1.0 mg/ml. Theminimum weighing is of 100 mg. Catecholamine standard curves are made upin 0.1 M perchloric acid/10⁻⁷ M ascorbic acid containing 50 ng/mldihydrobenzylamine. The highest standard contains 300 ng/ml dopamine,100 ng/ml dihydroxyphenylacetic acid, and 75 ng/ml HVA, and serialdilutions are made to 75%, 40%, 20%, and 10% of the highest standardmix. A 0% standard is also included for a total of 6 standards. Linearregression analysis of standard concentration vs. peak area is performedto determine analyte concentration in the experimental samples.Triplicate injections of each standard and sample are used and theresults from the triplicate injections are averaged. Calibration(standard) curves is run with each sample set to account for dailyvariation in the HPLC system. Calibration standards is run at thebeginning of a sample set and interspersed with the samples to determinevariation in the sampling method over the course of analysis of a sampleset. System suitability is checked daily by 5 replicate injections of acontrol sample spiked with dopamine, dihydroxyphenylacetic acid and HVA.Variations in peak area and retention time of less than 2% (forbiological samples) are considered acceptable.

Total numbers of Nissl- and TH-stained neurons throughout the entireSubstantia Nigra are counted stereologically in a blinded fashion withStereo Investigator software (MicroBrightfield, Williston, Vt.) usingthe Optical Fractionator probe module. Quantitation of striatal terminiis determined by TH immunostaining and digital image analysis (Scion,Frederick, Md.).

Behavioral Analysis: The behavioral tests are performed with 12 mice pergroup every week. The rest of the groups are administered drugs and donot undergo behavioral tests. Mice are monitored for rotarod performanceand pre-conditioned for 3 days prior to initiation of testing. In brief,mice are placed on a partitioned rotating rod (Rotamex Rota-rodapparatus, Columbus Instruments, Columbus, Ohio) and tested at a 5, 10,and 15 rpm for a maximum of 180 seconds at each speed with a minimum of5 minutes rest between attempts. Overall rotarod performance (ORP) iscalculated as the area under the curve using Prism (version 4, GraphpadSoftware, San Diego, Calif.) from the plot of the time that the animalremains on the rod and the function of the rotation speed. Learned motorskills in the rotarod beyond that of pre-conditioned skills are evidentfrom significant increases in ORP and/or diminished variances within thePBS control group. Grip strength is assessed by the paw grip endurance(PaGE) test. For PaGE analysis each mouse is placed on the wire-lid of aconventional housing cage and gently shaken to prompt the mouse to holdon to the grid. The lid is turned upside down and the duration until themouse release both hind limbs is determined. Each mouse is given threeattempts with a maximum duration of 90 seconds and the longest latencyrecorded.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A compound having the general formula I:

wherein: R₂ and R₃ are each 4-amino-butyryl (GABA) and R₁ is selectedfrom the group consisting of hydrogen and 4-amino-butyryl (GABA); and R₄is selected from the group consisting of hydrogen, alkyl,butyryloxyalkyl and 4-amino butyryloxyalkyl, or a pharmaceuticallyacceptable acid addition salt thereof.
 2. The compound of claim 1,wherein R₁ is hydrogen.
 3. The compound of claim 2, wherein R₄ is alkyl.4. The compound of claim 3, wherein said alkyl is butyl.
 5. The compoundof claim 4, being in a form of said a pharmaceutically acceptable acidaddition salt.
 6. The compound of claim 5, wherein said acid additionsalt is a benzenesulfonic acid addition salt (besylate).
 7. The compoundof claim 1, wherein each of R₁-R₃ is 4-amino-butyryl (GABA).
 8. Thecompound of claim 7, wherein R₄ is alkyl.
 9. The compound of claim 8,wherein said alkyl is butyl.
 10. The compound of claim 8, wherein saidalkyl is methyl.
 11. The compound of claim 1, wherein: R₁ and R₂ areeach 4-amino-butyryl (GABA); R₁ is hydrogen; and R₄ is butyl, or abenzenesulfonic acid addition salt (besylate) thereof.
 12. The compoundof claim 1, wherein: R₁, R₂ and R₃ are each 4-amino-butyryl (GABA); andR₄ is butyl.
 13. The compound of claim 1, wherein: R₁, R₂ and R₃ areeach 4-amino-butyryl (GABA); and R₄ is methyl.
 14. The compound of claim1, wherein said acid addition salt is selected from the group consistingof hydrochloric acid addition salt, acetic acid addition salt, ascorbicacid addition salt, benzenesulfonic acid addition salt (besylate),naphthylsulfonic acid addition salt (napsylate), camphorsulfonic acidaddition salt, citric acid addition salt, maleic acid addition salt,methanesulfonic acid addition salt (mesylate), oxalic acid additionsalt, phosphoric acid addition salt, succinic acid addition salt,sulfuric acid addition salt and tartaric acid addition salt.
 15. Apharmaceutical composition comprising, as an active ingredient, thecompound of claim 1 and a pharmaceutically acceptable carrier.
 16. Anarticle-of-manufacturing comprising the pharmaceutical composition ofclaim 15, the composition being packaged in a packaging material andidentified in print, on or in said packaging material, for use in thetreatment of a neurodegenerative disease or disorder.
 17. Thearticle-of-manufacturing of claim 16, wherein said neurodegenerativedisease or disorder is Parkinson's disease.
 18. A method of treating aneurodegenerative disease or disorder, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim 1, thereby treating theneurodegenerative disease or disorder disease.
 19. The method of claim18, wherein said neurodegenerative disease or disorder is Parkinson'sdisease.
 20. A pharmaceutical composition comprising, as an activeingredient, the compound of claim 11 and a pharmaceutically acceptablecarrier.
 21. An article-of-manufacturing comprising the pharmaceuticalcomposition of claim 20, the composition being packaged in a packagingmaterial and identified in print, on or in said packaging material, foruse in the treatment of a neurodegenerative disease or disorder.
 22. Thearticle-of-manufacturing of claim 21, wherein said neurodegenerativedisease or disorder is Parkinson's disease.
 23. A method of treating aneurodegenerative disease or disorder, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim 11, thereby treating theneurodegenerative disease or disorder disease.
 24. The method of claim23, wherein said neurodegenerative disease or disorder is Parkinson'sdisease.
 25. A pharmaceutical composition comprising, as an activeingredient, the compound of claim 12 and a pharmaceutically acceptablecarrier.
 26. An article-of-manufacturing comprising the pharmaceuticalcomposition of claim 12, the composition being packaged in a packagingmaterial and identified in print, on or in said packaging material, foruse in the treatment of a neurodegenerative disease or disorder.
 27. Thearticle-of-manufacturing of claim 26, wherein said neurodegenerativedisease or disorder is Parkinson's disease.
 28. A method of treating aneurodegenerative disease or disorder, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim 12, thereby treating theneurodegenerative disease or disorder disease.
 29. The method of claim28, wherein said neurodegenerative disease or disorder is Parkinson'sdisease.
 30. A pharmaceutical composition comprising, as an activeingredient, the compound of claim 13 and a pharmaceutically acceptablecarrier.
 31. An article-of-manufacturing comprising the pharmaceuticalcomposition of claim 30, the composition being packaged in a packagingmaterial and identified in print, on or in said packaging material, foruse in the treatment of a neurodegenerative disease or disorder.
 32. Thearticle-of-manufacturing of claim 31, wherein said neurodegenerativedisease or disorder is Parkinson's disease.
 33. A method of treating aneurodegenerative disease or disorder, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim 13, thereby treating theneurodegenerative disease or disorder disease.
 34. The method of claim33, wherein said neurodegenerative disease or disorder is Parkinson'sdisease.